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ABSTRACT: BACKGROUND: Resveratrol increases lifespan and decreases the risk of many cancers. We hypothesized resveratrol will slow the growth of human prostate cancer xenografts. METHODS: 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 mm(3) . 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. RESULTS: 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. CONCLUSION: 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. Prostate © 2012 Wiley Periodicals, Inc.
The Prostate 11/2012; · 3.48 Impact Factor
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ABSTRACT: The voltage-dependent anion channel (VDAC), a major pore-forming protein in the outer membrane of mitochondria, is also found in the plasma membrane of a large number of cells, where in addition to its role in regulating cellular ATP release and volume control, it is important for maintaining redox homeostasis. Cell surface VDAC is a receptor for plasminogen kringle 5, which promotes partial closure of the channel. In this study, we demonstrate that VDAC binds tissue-type plasminogen activator (t-PA) on human neuroblastoma SK-N-SH cells. Binding of t-PA to VDAC induces a decrease in Km and an increase in the Vmax for activation of its substrate, plasminogen (Pg). This results in accelerated Pg activation when VDAC, t-PA and Pg are bound together. VDAC is also a substrate for plasmin, hence, it mimics fibrin activity. Binding of t-PA to VDAC occurs between a t-PA fibronectin type I finger domain located between amino acids I5-N37 and a VDAC region between amino acids G20YGFG24. These VDAC residues correspond to a GxxxG repeat motif commonly found in amyloid β-peptides, which is necessary for aggregation when these peptides form fibrillar deposits on the cell surface. Furthermore, we also show that Pg kringle 5 is a substrate for the NADH-dependent reductase activity of VDAC. This ternary complex is an efficient proteolytic complex that may facilitate removal of amyloid β-peptide deposits from the normal brain and cell debris from injured brain tissue.
Journal of Biological Chemistry 11/2012; · 4.77 Impact Factor
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ABSTRACT: BACKGROUND: No- and low-carbohydrate diets delay tumor growth compared to western diet (WD) in prostate cancer (PCa) xenograft studies. The effect of these diets in concert with androgen deprivation is unknown. METHODS: A total of 160 male SCID mice were injected with 1× 10(5) LAPC-4 human PCa cells. Of these, 150 mice were castrated and randomized to an ad libitum WD or fed via a paired-feeding protocol with a no-carbohydrate ketogenic diet (NCKD), 10% carbohydrate diet, or 20% carbohydrate diet. The remaining 10 mice were not castrated and were fed an ad libitum WD. The mice were sacrificed once volumes reached 1,000 mm(3) and survival tested using the log-rank test. Serum from the median surviving 8 mice/group was assayed for insulin, IGF-1, and IGFBP-3. RESULTS: Body weights were roughly equal among groups. The 10 non-castrated mice experienced accelerated tumor growth. Among castrated mice, WD had the most rapid tumor growth; 20% carbohydrate diet the slowest (P = 0.046). Survival was not significantly different among the various carbohydrate restricted groups (P = 0.51). When pooled, there was a non-significant trend (P = 0.11) in improved survival among the carbohydrate restricted diets versus WD. No significant difference in serum insulin, IGF-1, and IGFBP-3 levels was noted among all groups at pre-randomization or at sacrifice. CONCLUSIONS: A 20% carbohydrate diet slowed tumor growth versus a WD. Though the benefit of carbohydrate restriction was somewhat less than in prior studies in non-castrate mice, these data still suggest diets achievable in humans may play a role in PCa management. Prostate © 2012 Wiley Periodicals, Inc.
The Prostate 10/2012; · 3.48 Impact Factor
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ABSTRACT: GRP78, a molecular chaperone with critical endoplasmic reticulum functions, is aberrantly expressed on the surface of cancer cells, including prostate and melanoma. Here it functions as a pro-proliferative and anti-apoptotic signaling receptor via NH(2)-terminal domain ligation. Auto-antibodies to this domain may appear in cancer patient serum where they are a poor prognostic indicator. Conversely, GRP78 COOH-terminal domain ligation is pro-apoptotic and anti-proliferative. There is no method to disrupt cell-surface GRP78 without compromising the total GRP78 pool, making it difficult to study cell-surface GRP78 function. We studied six cell lines representing three cancer types. One cell line per group expresses high levels of cell-surface GRP78, and the other expresses low levels (human hepatoma: Hep3B and HepG2; human prostate cancer: PC3 and 1-LN; murine melanoma: B16F0 and B16F1). We investigated the effect of Escherichia coli subtilase cytoxin catalytic subunit (SubA) on GRP78. We report that SubA specifically cleaves cell-surface GRP78 on HepG2, 1-LN, and B16F1 cells without affecting intracellular GRP78. B16F0 cells (GRP78(low)) have lower amounts of cleaved cell-surface GRP78. SubA has no effect on Hep3B and PC3 cells. The predicted 28-kDa GRP78 COOH-terminal fragment is released into the culture medium by SubA treatment, and COOH-terminal domain signal transduction is abrogated, whereas pro-proliferative signaling mediated through NH(2)-terminal domain ligation is unaffected. These experiments clarify cell-surface GRP78 topology and demonstrate that the COOH-terminal domain is necessary for pro-apoptotic signal transduction occurring upon COOH-terminal antibody ligation. SubA is a powerful tool to specifically probe the functions of cell-surface GRP78.
Journal of Biological Chemistry 07/2012; 287(39):32755-69. · 4.77 Impact Factor
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ABSTRACT: The HSP70 family member GRP78 is a selective tumor marker upregulated on the surface of many tumor cell types, including melanoma, where it acts as a growth factor receptor-like protein. Receptor-recognized forms of the proteinase inhibitor α2-macroglobulin (α2M*) are the best-characterized ligands for GRP78, but in melanoma and other cancer patients, autoantibodies arise against the NH2-terminal domain of GRP78 that react with tumor cell-surface GRP78. This causes the activation of signaling cascades that are proproliferative and antiapoptotic. Antibodies directed against the COOH-terminal domain of GRP78, however, upregulate p53-mediated proapoptotic signaling, leading to cell death. Here, we describe the binding characteristics, cell signaling properties, and downstream cellular effects of three novel murine monoclonal antibodies. The NH2-terminal domain-reactive antibody, N88, mimics α2M* as a ligand and drives PI 3-kinase-dependent activation of Akt and the subsequent stimulation of cellular proliferation in vitro. The COOH-terminal domain-reactive antibody, C38, acts as an antagonist of both α2M* and N88, whereas another, C107, directly induces apoptosis in vitro. In a murine B16F1 melanoma flank tumor model, we demonstrate the acceleration of tumor growth by treatment with N88, whereas C107 significantly slowed tumor growth whether administered before (P<0.005) or after (P<0.05) tumor implantation.
Melanoma research 04/2012; 22(3):225-35. · 2.06 Impact Factor
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ABSTRACT: What's known on the subject? and What does the study add? It is known that both lactate inhibition and carbohydrate restriction inhibit tumour growth. What is unknown is whether the two work synergistically together. This study adds that though the combination of lactate inhibition and carbohydrate restriction did not synergistically slow tumour growth in our model, we confirmed that carbohydrate restriction started after tumour inoculation slowed tumour growth. Moreover, lactate inhibition resulted in changes in the tumour microenvironment that may have implications for future metabolic targeting of prostate cancer growth.
• 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 × 10(5) 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.
BJU International 03/2012; 110(7):1062-9. · 2.84 Impact Factor
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ABSTRACT: 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 (NaIO(4))-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.
PLoS ONE 01/2012; 7(12):e52693. · 4.09 Impact Factor
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ABSTRACT: Tetrameric α(2)-macroglobulin (α(2)M), 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 α(2)M (α(2)M*) binds to cancer cell surface GRP78 and triggers proliferative and antiapoptotic signaling. We have studied the role of α(2)M* 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 α(2)M* promotes phosphorylation of mTOR, TSC2, S6-Kinase, 4EBP, Akt(T308), and Akt(S473) in a concentration and time-dependent manner. Rheb, Raptor, and Rictor also increased. α(2)M* 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 α(2)M*-induced S6-Kinase phosphorylation at T389 and kinase activity in Raptor immunoprecipitates. α(2)M*-treated cells demonstrate about a twofold increase in mTORC2 kinase activity as determined by kinase assay of Akt(S473) phosphorylation and levels of p-Akt(S473) 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 α(2)M*-induced phosphorylation of Akt(S473) phosphorylation in Rictor immunoprecipitates.
Binding of α(2)M* to prostate cancer cell surface GRP78 upregulates mTORC1 and mTORC2 activation and promotes protein synthesis in the prostate cancer cells.
PLoS ONE 01/2012; 7(12):e51735. · 4.09 Impact Factor
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ABSTRACT: 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 immunoglobulin (Ig) G autoantibodies at these stages and evaluated their capacities to affect the protein B-dependent protein kinase 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 through protein B-dependent protein kinase signaling pathways. They also mimic the effects of α2-macroglobulin on the upregulation of GRP78 and X-box binding protein 1, activating transcription factor 6 α, and serine/threonine-protein kinase/endoribonuclease precursor α as endoplasmic reticulum stress biomarkers and show no effect on expression or activation of caspases 3, 9, or 12. In conclusion, the anti-GRP78 IgG autoantibodies downregulate apoptosis and activate unfolded protein response mechanisms, which are essential to promote melanoma cell growth and survival.
Melanoma research 05/2011; 21(4):323-34. · 2.06 Impact Factor
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ABSTRACT: GRP78, a well characterized chaperone in the endoplasmic reticulum, is critical to the unfolded protein response. More recently,
it has been identified on the cell surface, where it has many roles. On cancer cells, it functions as a signaling receptor
coupled to proproliferative/antiapoptotic and promigratory mechanisms. In the current study, we demonstrate that ligation
of prostate cancer cell surface GRP78 by its natural ligand, activated α2-macroglobulin (α2M*), results in a 2–3-fold up-regulation in the synthesis of prostate-specific antigen (PSA). The PSA is secreted into the
medium as an active proteinase, where it binds to native α2M. The resultant α2M·PSA complexes bind to GRP78, causing a 1.5–2-fold increase in the activation of MEK1/2, ERK1/2, S6K, and Akt, which is coupled
with a 2–3-fold increase in DNA and protein synthesis. PSA is a marker for the progression of prostate cancer, but its mechanistic
role in the disease is unclear. The present studies suggest that PSA may be involved in a signal transduction-dependent feedback
loop, whereby it promotes a more aggressive behavior by human prostate cancer cells.
Journal of Biological Chemistry 01/2011; 286(2):1248-1259. · 4.77 Impact Factor
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ABSTRACT: Scarring is a highly prevalent and multifactorial process, yet no studies to date have attempted to distinguish pathologic from nonpathologic scarring.
This article defines and proposes methods of classifying pathologic scarring as it pertains to clinical presentation.
The authors propose a new scar scale that incorporates pain and functional impairment.
The modified Patient and Observer 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 scale has great potential in evaluating patient response to treatment and analyzing clinical outcomes.
Plastic and reconstructive surgery 01/2011; 127(1):242-7. · 2.74 Impact Factor
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ABSTRACT: The unfolded protein response (UPR) is an adaptive survival mechanism through which cells can weather the stress of misfolded protein accumulation induced by a wide variety of pathophysiologic and pharmacologic insults. The ER chaperone GRP78 is a central modulator of the UPR both through its protein-binding capacity and its direct regulation of the UPR signaling molecules IRE1α, PERK, and ATF6. Recent reports have revealed the presence of GRP78 on the surface of cancer cells. Biological roles for cell-surface GRP78 include competing NH(2)-domain and COOH-domain agonist receptor activities that induce opposite effects on proliferation and apoptosis. Modulation of the UPR impacts both of these processes directly and indirectly. Here, we outline methods that we use to investigate UPR modulation via direct ligation of cell-surface GRP78. Specifically, we review methods of cell culture, cell-signaling analysis with emphasis on UPR components, and ultimately, the impact that these have on cell proliferation, survival, and apoptosis.
Methods in enzymology 01/2011; 489:245-57. · 1.90 Impact Factor
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ABSTRACT: 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 immunoglobulin Gs are merely a biomarker, or whether they actually promote the 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 used the antisera from these mice for in-vitro cell signaling and proliferation assays. The immunodominant epitope in patients with cancer was well represented in the antibody repertoire of these immunized mice. We observed significantly accelerated tumor growth, and shortened survival in GRP78-immunized mice compared with controls. Furthermore, antisera from these mice, and purified anti-GRP78 immunoglobulin G from similarly immunized mice, stimulate Akt phosphorylation and proliferation in B16F1 and human DM6 melanoma cells in culture. These studies show 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. As GRP78 is present on the surface of many types of cancer cells, this hypothesis has broad clinical and therapeutic implications.
Melanoma research 12/2010; · 2.06 Impact Factor
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ABSTRACT: GRP94 (gp96)-peptide complexes can be internalized by APCs and their associated peptides cross-presented to yield activation of CD8(+) T cells. Investigations into the identity (or identities) of GRP94 surface receptors have yielded conflicting results, particularly with respect to CD91 (LRP1), which has been proposed to be essential for GRP94 recognition and uptake. To assess CD91 function in GRP94 surface binding and endocytosis, these parameters were examined in mouse embryonic fibroblast (MEF) cell lines whose expression of CD91 was either reduced via RNA interference or eliminated by genetic disruption of the CD91 locus. Reduction or loss of CD91 expression abrogated the binding and uptake of receptor-associated protein, an established CD91 ligand. Surface binding and uptake of an N-terminal domain of GRP94 (GRP94.NTD) was unaffected. GRP94.NTD surface binding was markedly suppressed after treatment of MEF cell lines with heparin, sodium chlorate, or heparinase II, demonstrating that heparin sulfate proteoglycans can function in GRP94.NTD surface binding. The role of CD91 in the cross-presentation 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 the CD91 ligands receptor-associated protein or activated α(2)-macroglobulin and occurred primarily via a fluid-phase, rather than receptor-mediated, uptake pathway. These data clarify conflicting data on CD91 function in GRP94 surface binding, endocytosis, and peptide cross-presentation and identify a role for heparin sulfate proteoglycans in GRP94 surface binding.
The Journal of Immunology 11/2010; 185(11):6819-30. · 5.79 Impact Factor
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ABSTRACT: GRP78, a well characterized chaperone in the endoplasmic reticulum, is critical to the unfolded protein response. More recently, it has been identified on the cell surface, where it has many roles. On cancer cells, it functions as a signaling receptor coupled to proproliferative/antiapoptotic and promigratory mechanisms. In the current study, we demonstrate that ligation of prostate cancer cell surface GRP78 by its natural ligand, activated α(2)-macroglobulin (α(2)M*), results in a 2-3-fold up-regulation in the synthesis of prostate-specific antigen (PSA). The PSA is secreted into the medium as an active proteinase, where it binds to native α(2)M. The resultant α(2)M·PSA complexes bind to GRP78, causing a 1.5-2-fold increase in the activation of MEK1/2, ERK1/2, S6K, and Akt, which is coupled with a 2-3-fold increase in DNA and protein synthesis. PSA is a marker for the progression of prostate cancer, but its mechanistic role in the disease is unclear. The present studies suggest that PSA may be involved in a signal transduction-dependent feedback loop, whereby it promotes a more aggressive behavior by human prostate cancer cells.
Journal of Biological Chemistry 11/2010; 286(2):1248-59. · 4.77 Impact Factor
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ABSTRACT: 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.
Journal of neuroimmunology 10/2010; 227(1-2):153-61. · 2.84 Impact Factor
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Ali A Al-Hashimi,
Jennifer Caldwell,
Mario Gonzalez-Gronow, Salvatore V Pizzo,
Danya Aboumrad,
Lindsay Pozza,
Hiam Al-Bayati,
Jeffrey I Weitz,
Alan Stafford,
Howard Chan,
Anil Kapoor,
Donald W Jacobsen,
Jeffrey G Dickhout,
Richard C Austin
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ABSTRACT: The increased risk of venous thromboembolism in cancer patients has been attributed to enhanced tissue factor (TF) procoagulant activity (PCA) on the surface of cancer cells. Recent studies have shown that TF PCA can be modulated by GRP78, an endoplasmic reticulum (ER)-resident molecular chaperone. In this study, we investigated the role of cell surface GRP78 in modulating TF PCA in several human cancer cell lines. Although both GRP78 and TF are present on the cell surface of cancer cells, there was no evidence of a stable interaction between recombinant human GRP78 and TF, nor was there any effect of exogenously added recombinant GRP78 on cell surface TF PCA. Treatment of cells with the ER stress-inducing agent thapsigargin, an inhibitor of the sarco(endo)plasmic reticulum Ca(2+) pump that causes Ca(2+) efflux from ER stores, increased cytosolic [Ca(2+)] and induced TF PCA. Consistent with these findings, anti-GRP78 autoantibodies that were isolated from the serum of patients with prostate cancer and bind to a specific N-terminal epitope (Leu(98)-Leu(115)) on cell surface GRP78, caused a dose-dependent increase in cytosolic [Ca(2+)] and enhanced TF PCA. The ability to interfere with cell surface GRP78 binding, block phospholipase C activity, sequester ER Ca(2+), or prevent plasma membrane phosphatidylserine exposure resulted in a significant decrease in the TF PCA induced by anti-GRP78 autoantibodies. Taken together, these findings provide evidence that engagement of the anti-GRP78 autoantibodies with cell surface GRP78 increases TF PCA through a mechanism that involves the release of Ca(2+) from ER stores. Furthermore, blocking GRP78 signaling on the surface of cancer cells attenuates TF PCA and has the potential to reduce the risk of cancer-related venous thromboembolism.
Journal of Biological Chemistry 09/2010; 285(37):28912-23. · 4.77 Impact Factor
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ABSTRACT: 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 mm(3). 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).
Cancer Prevention Research 09/2010; 3(9):1124-31. · 4.91 Impact Factor
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ABSTRACT: Since the presence of F1F0 adenosine triphosphate (ATP) synthase was discovered on the surface of human cells, numerous studies have elucidated new
functions for this molecule that classically functions as part of the electron transport chain in mitochondria. Like mitochondrial
ATP synthase, the cell surface form of this molecule functions as an energy-producing proton pump, coupling the production
of extracellular ATP with the transport of protons outside the cell. ATP synthase also acts as an endothelial cell surface
receptor for angiostatin, which has led several groups to examine this molecule as a target for inhibiting angiogenesis. Given
its involvement in proton transport and the pH-dependent activity of angiostatin and antibodies directed against it, ATP synthase
likely functions in part to regulate pH for endothelial cells existing in an acidic microenvironment. In addition, it catalyzes
the synthesis and hydrolysis of ATP in the extracellular milieu, potentially affecting purinergic signaling. ATP synthase
also plays a role in endothelial cell signaling in response to flow-induced shear stress. Recently, ATP synthase on the surface
of endothelial cells has been identified as the receptor for two additional proteins: coupling factor 6 and endothelial monocyte-activating
polypeptide II. This chapter will focus on the role of endothelial cell surface ATP synthase in angiogenesis, shear stress
response, and as a receptor.
KeywordsCell surface ATP synthase-Alpha subunit of ATP synthase-Beta subunit of ATP synthase-Endothelial cell-Angiostatin-Angiogenesis-Anti-angiogenic treatment-ATP synthesis-ATP hydrolysis-Purinergic signaling-pH regulation-Coupling factor 6-Shear stress-Endothelial monocyte-activating polypeptide II-Heparan sulfate-Caveolae-Sangivamycin-Monoclonal antibody therapy
04/2010: pages 139-159;
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ABSTRACT: We have previously shown that treatment of prostate cancer and melanoma cells expressing GRP78 on their cell surface with antibody directed against the COOH-terminal domain of GRP78 upregulates and activates p53 causing decreased cell proliferation and upregulated apoptosis. In this report, we demonstrate that treatment of 1-LN prostate cancer cells with this antibody decreases cell surface expression of GRP78, Akt(Thr308) and Akt(Ser473) kinase activities and reduces phosphorylation of FOXO, and GSK3beta. This treatment also suppresses activation of ERK1/2, p38 MAPK and MKK3/6; however, it upregulates MKK4 activity. JNK, as determined by its phosphorylation state, is subsequently activated, triggering apoptosis. Incubation of cells with antibody reduced levels of anti-apoptotic Bcl-2, while elevating pro-apoptotic BAD, BAX and BAK expression as well as cleaved caspases-3, -7, -8 and -9. Silencing GRP78 or p53 gene expression by RNAi prior to antibody treatment abrogated these effects. We conclude that antibody directed against the COOH-terminal domain of GRP78 may prove useful as a pan suppressor of proliferative/survival signaling in cancer cells expressing GRP78 on their cell surface.
Cancer biology & therapy 01/2010; 9(2):142-52. · 2.64 Impact Factor
