Aditi Banerjee

Cag pathogenicity island-independent up-regulation of matrix metalloproteinases-9 and -2 secretion and expression in mice by Helicobacter pylori infection.

Helicobacter pylori cag pathogenicity island (PAI) is a major determinant of gastric injury via induction of several matrix metalloproteinases (MMPs). In the present study, we examined the influence of the cag PAI on gastric infection and MMP-9 production in mice and in cultured cells. A new mouse colonizing Indian H. pylori strain (AM1) that lacks the cag PAI was used to study the cag PAI importance in inflammation. Groups of C57BL/6 mice were inoculated separately with H. pylori strains AM1 and SS1 (cag+), gastric tissues were histologically examined, and bacterial colonization was scored by quantitative culture. Mice infected with either cag+ or cag- H. pylori strains showed gastric inflammation and elevated MMP-3 production. Significant up-regulation of pro-MMP-9 secretion and gene expression in H. pylori infected gastric tissues indicate dispensability of cag PAI for increased pro-MMP-9 secretion and synthesis in mice. In agreement, cell culture studies revealed that both AM1 and SS1 were equipotent in pro-MMP-9 induction in human gastric epithelial cells. Both strains showed moderate increase in MMP-2 activity in vivo and in vitro. In addition, increased secretion of tumor necrosis factor (TNF)-α, interleukin (IL)-1β, and IL-6 induced pro-MMP-9 secretion and synthesis in AM1 or SS1 strain-infected mice suggesting elicitation of pro-inflammatory cytokines by both cag- and cag+ genotype. Moreover, tissue inhibitors of metalloproteinase-1 expression were decreased with increase in pro-MMP-9 induction. These data show that H. pylori may act through different pathways other than cag PAI-mediated for gastric inflammation and contribute to up-regulation of MMP-9 via pro-inflammatory cytokines.

Unfolded protein response is required in nu/nu mice microvasculature for treating breast tumor with tunicamycin.

Up-regulation of the dolichol pathway, a “hallmark” of asparagine-linked protein glycosylation, enhances angiogenesis in vitro. The dynamic relationship between these two processes is now evaluated with tunicamycin. Capillary endothelial cells treated with tunicamycin were growth inhibited and could not be reversed with exogenous VEGF165. Inhibition of angiogenesis is supported by down-regulation of (i) phosphorylated VEGFR1 and VEGFR2 receptors; (ii) VEGF165-specific phosphotyrosine kinase activity; and (iii) MatrigelTM invasion and chemotaxis. In vivo, tunicamycin prevented the vessel development in MatrigelTM implants in athymic Balb/c (nu/nu) mice. Immunohistochemical analysis of CD34 (p < 0.001) and CD144 (p < 0.001) exhibited reduced vascularization. A 3.8-fold increased expression of TSP-1, an endogenous angiogenesis inhibitor in MatrigelTM implants correlated with that in tunicamycin (32 h)-treated capillary endothelial cells. Intravenous injection of tunicamycin (0.5 mg/kg to 1.0 mg/kg) per week slowed down a double negative (MDA-MB-435) grade III breast adenocarcinoma growth by ∼50–60% in 3 weeks. Histopathological analysis of the paraffin sections indicated significant reduction in vessel size, the microvascular density and tumor mitotic index. Ki-67 and VEGF expression in tumor tissue were also reduced. A significant reduction of N-glycan expression in tumor microvessel was also observed. High expression of GRP-78 in CD144-positive cells supported unfolded protein response-mediated ER stress in tumor microvasculature. ∼65% reduction of a triple negative (MDA-MB-231) breast tumor xenograft in 1 week with tunicamycin (0.25 mg/kg) given orally and the absence of systemic and/or organ failure strongly supported tunicamycins potential for a powerful glycotherapeutic treatment of breast cancer in the clinic.

Leishmanial lipid suppresses tumor necrosis factor α, interleukin-1β, and nitric oxide production by adherent synovial fluid mononuclear cells in rheumatoid arthritis patients and induces apoptosis through the mitochondrial-mediated pathway

OBJECTIVE Leishmanial lipid is a strong immunosuppressor of host cells. Inhibition of the inflammatory responses of synovial cells through induction of apoptosis is one of the main targets of therapeutic intervention in rheumatoid arthritis (RA). This study was undertaken to examine the antiinflammatory and apoptosis-inducing effects of leishmanial lipid on adherent synovial fluid mononuclear cells (SFMCs) in patients with RA. METHODS Lipid was extracted from a Leishmania donovani promastigote (MHO/IN/1978/UR6) by the Bligh and Dyer method. Nitric oxide (NO) was measured using the Griess reaction, and enzyme-linked immunosorbent assays for cytokines, NF-kappaB, and cytochrome c were performed. Levels of cytokines, inducible nitric oxide synthase, caspases, Bcl-2, Bax, t-Bid, and cytochrome c in the cell lysate and of NF-kappaB p65 in the nucleus were determined by Western blotting. Microscopic analysis, nuclear staining, DNA fragmentation assay, fluorescence-activated cell sorting, colorimetric assay for caspases, and fluorescent probe for measurement of mitochondrial membrane potential were used to study the leishmanial lipid-induced apoptotic pathway in SFMCs. RESULTS Leishmanial lipid inhibited the release of tumor necrosis factor alpha, interleukin-1beta, and NO in the culture, decreased their cytosolic protein levels, and decreased NF-kappaB p65 levels in SFMCs, in a dose-dependent manner. It had the reverse effect on interleukin-10 levels. Leishmanial lipid-induced apoptosis involved the activation of caspase 3, caspase 9, and Bax, the release of cytochrome c, the alteration of mitochondrial membrane potential, and the down-regulation of Bcl-2. CONCLUSION These results suggest that leishmanial lipid has strong antiinflammatory and apoptosis-inducing effects on SFMCs from patients with RA, and that apoptosis occurs via the mitochondrial pathway.

Mannosylphosphodolichol synthase overexpression supports angiogenesis

Abstract Mannosylphosphodolichol synthase (DPMS) plays a critical role in Glc3Man9GlcNAc2-PP-Dol (lipid-linked oligosaccha-ride, LLO) biosynthesis, an essential intermediate in asparagine-linked (N-linked) protein glycosylation. We observed earlier that phosphorylation of DPMS increases the catalytic activity of the enzyme by increasing the Vmax as well as the enzyme turnover (kcat) without significantly changing the Km for GDP-mannose. As a result, LLO biosynthesis, turnover and protein N-glycosylation are increased. This is manifested in increased proliferation of capillary endothelial cells, i.e. angiogenesis. We have then asked, if the phosphorylation event or the upregulation of DPMS due to overproduction of the enzyme is the key factor in upregulating angiogenesis? This question has been answered by isolating a stable capillary endothelial cell clone overexpressing the gene encoding DPMS. Our results indicate that the DPMS-overexpressing clone has a high level of DPMS mRNA as judged by QRT-PCR. The clone also expresses nearly four times more DPMS protein than the clone transfected with pEGFP-N1 vector only (i.e. control) as analyzed by Western blotting. Most importantly, the overexpressing DPMS clone has ∼108% higher DPMS activity than the vector control. Immunofluorescence microscopy with Texas Red-conjugated wheat germ agglutinin indicates a high level of expression of (GlcNAc-β-(1,4)-GlcNAc) 1-4-β-GlcNAc-NeuAc glycans on the external surface of the capillary endothelial cells overexpressing DPMS. Increased cellular proliferation and accelerated healing of the wound induced by mechanical stress of the DPMS-overexpressing clone unequivocally supports a role of DPMS in angiogenesis.

N-Acetylglucosaminyl 1-Phosphate Transferase: An Excellent Target for Developing New Generation Breast Cancer Therapeutic

Studies from our laboratory have explained that breast tumor progression can be attenuated by targeting the N-linked glycoproteins of the tumor microvasculature and that of tumor cells alike with a protein N-glycosylation inhibitor, tunicamycin. Absence of N-glycosylation leads to an accumulation of un- or mis-folded proteins in the ER and the cell develops “ER stress”. The result is cell cycle arrest, and induction of apoptosis mediated by unfolded protein response (upr) signaling. Tunicamycin inhibited in vitro and in vivo (Matrigel™ implants in athymic nude mice) angiogenesis in a dose dependent manner. The action is irreversible and survived under tumor microenvironment, i.e., in the presence of FGF-2 or VEGF or higher serum concentration. Importantly, tunicamycin prevented the progression of double negative (ER−/PR−/Her2+) and triple negative (ER−/PR−/Her2−) breast tumors by ~55 to 65 % in 3 weeks in athymic nude mice [Balb/c(nu/nu)]. Analyses of paraffin sections exhibited “ER stress” in both microvasculature and in tumor tissue.

Balancing life with glycoconjugates: monitoring unfolded protein response-mediated anti-angiogenic action of tunicamycin by Raman Spectroscopy

Asparagine-linked protein glycosylation is a hallmark for glycoprotein structure and function. Its impairment by tunicamycin [a competitive inhibitor of N-acetylglucos-aminyl 1-phosphate transferase (GPT)] has been known to inhibit neo-vascularization (i.e., angiogenesis) in humanized breast tumor due to an induction of endoplasmic reticulum (ER) stress-mediated unfolded protein response (UPR). The studies presented here demonstrate that (i) tunicamycin inhibits capillary endothelial cell proliferation in a dose-dependent manner; (ii) treated cells are incapable of forming colonies upon its withdrawal; and (iii) tunicamycin treatment causes nuclear fragmentation. Tunicamycin-induced ER stress-mediated UPR event in these cells was studied with the aid of Raman spectroscopy, in particular, the interpretation of bands at 1672, 1684, and 1694 cm–1, which are characteristics of proteins and originate from C=O stretching vibrations of mono-substituted amides. In tunicamycin-treated cells, these bands decreased in area as follows: at 1672 cm–1 by 41.85 % at 3 h and 55.39 % at 12 h; at 1684 cm–1 by 20.63 % at 3 h and 40.08 % at 12 h; and also at 1994 cm–1 by 33.33 % at 3 h and 32.92 % at 12 h, respectively. Thus, in the presence of tunicamycin, newly synthesized protein chains fail to arrange properly into their final secondary and/or tertiary structures, and the random coils they form had undergone further degradation.

Importance of a Factor VIIIc-Like Glycoprotein Expressed in Capillary Endothelial Cells (eFactor VIIIc) in Angiogenesis

Factor VIII is a large, 2,332-residue plasma glycoprotein that acts as a regulatory cofactor in the process of blood coagulation [1–3]. It binds to activated factor IX (factor IXa) in the presence of calcium and negatively charged phospholipids at the surface of activated platelets to form a membrane-associated, proteolytically active complex. Upon complex formation, the V max of factor IXa is increased by approximately 200,000-fold, promoting the rapid activation of its substrate, the serine protease factor X. The proteolytic conversion of factor X to its active form, factor Xa, is a central control point in the coagulation cascade, leading to activation of thrombin, formation of a fibrin mesh, and establishment of a stable blood clot. The binding of factor VIIIc and other activated proteins to these membrane surfaces allows for localization of the procoagulation process to sites of vascular damage.

Abstract 590: Andrographolide suppresses prostate cancer cell migration and alters the expression of vimentin, ZO-1 and MMP-11

Proceedings: AACR 103rd Annual Meeting 2012‐‐ Mar 31‐Apr 4, 2012; Chicago, IL Prostate cancer is the most common cancer and the second cause of cancer-related deaths in males in the United States. The development of novel chemopreventive strategies may be extremely helpful to prevent and arrest the development of this neoplasm. Andrographolide, a labdane diterpenoid that is the main bioactive component of the medicinal plant Andrographis paniculata, has anti-inflammatory and anticarcinogenic properties. In this study, we sought to examine the effect of andrographolide on PC3 and LNCaP prostate cancer epithelial cells. Andrographolide significantly inhibited PC3 cell growth at a concentration of 10µM after 24 h of treatment (P < 0.001), and inhibited LNCaP cell growth at a concentration of 15 µM after 24 h of treatment (P < 0.001). Wound healing assay and boyden chamber experiments were performed in order to determine whether andrographolide had an effect on migration and invasion. When PC3 and LNCaP cells were treated with andrographolide the total number of migrating and invading cells was significantly reduced (P < 0.001) when compared to control. To study possible anti-migration and anti-invasion mechanisms of andrographolide on PC3 and LNCap cells, we examined the expression of various adhesion molecules. Andrograholide significantly decreased the expression of vimentin in PC3 and LNCap treated cells. However, PC3 and LNCaP cells treated with andrographolide had increased expression levels of occludin and ZO-1. In addition, immunofluorescence and western blot analysis showed that PC3 and LNCaP cells treated with andrographolide had decresased expression levels of MMP-11. Our results suggest that the anti-migration and anti-invasion effects of andrographolide in PC3 and LNCaP cells may be associated with alterations in the expression of occluding, ZO-1 and MMP-11. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 590. doi:1538-7445.AM2012-590

Tunicamycin-induced ER stress in breast cancer cells neither expresses GRP78 on the surface nor secretes it into the media

GRP78 (an Mr 78 kDa calcium dependent glucose binding protein) is located in ER lumen. It functions as ER chaperone and translocates proteins for glycosylation at the asparagine residue present in the sequon Asn-X-Ser/Thr. Paraffin sections from N-glycosylation inhibitor tunicamycin treated ER-/PR-/HER2+ (double negative) breast tumor in athymic nude mice exhibited reduced N-glycan but increased GRP78 expression. We have evaluated the effect of tunicamycin on cellular localization of GRP78 in metastatic human breast cancer cells MDA-MB-231 (ER-/PR-/HER2-). Tunicamycin inhibited cell proliferation in a time and dose-dependent manner. Nonmetastatic estrogen receptor positive (ER+) MCF-7 breast cancer cells were also equally effective. GRP78 expression (protein and mRNA) was higher in tunicamycin (1.0 μg/mL) treated MCF-7 and MDA-MB-231 cells. GRP78 is an ER stress marker, so we have followed its intracellular localization using immunofluorescence microscopy after subjecting the cancer cells to various stress conditions. Unfixed cells stained with either FITC-conjugated Concanavalin A (Con A) or Texas-red conjugated wheat germ agglutinin (WGA) exhibited surface expression of N-glycans but not GRP78. GRP78 became detectable only after a brief exposure of cells to ice-cold methanol. Western blotting did not detect GRP78 in conditioned media of cancer cells whereas it did for MMP-1. The conclusion, GRP78 is expressed neither on the outer-leaflet of the (ER-/PR-/HER2-) human breast cancer cells nor it is secreted into the culture media during tunicamycin-induced ER stress. Our study therefore suggests strongly that anti-tumorigenic action of tunicamycin can be modeled to develop next generation cancer therapy, i.e., glycotherapy for treating breast and other sold tumors.

Abstract 4509: GRP78 isneitherexpressed on ER-/PR-/Her2-human breast cancer cell surfacenorsecreted in the culture media

GRP78 (Mr78 kDa) is a glucose regulated protein. It is a member of the heat-shock protein (HSP) family but located in the lumen of the endoplasmic reticulum (ER). Its function as ER chaperone translocating protein across the ER membrane that needs to be glycosylated at the asparagine residue present in the sequeon Asn-X-Ser/Thr (N-linked glycosylation) is well recognized. This important biochemical event is essential for glycoprotein folding and function. For example, when the protein N-glycosylation is impaired with tunicamycin a protein N-glycosylation inhibitor, angiogenesis, a hallmark for tumor progression and metastasis is inhibited due to an induction of ER stress. Under such condition, GRP78 is overexpressed in microvasculature and in tumor tissue, concluding that GRP78 is a master regulator in ER stress induced unfolded protein response (upr)-mediated apoptosis in tumor microvasculature (J. Biol. Chem. 286, 29127-29138, 2011; Pure Appl. Chem. 84, 1907-1918, 2012). This contradicts the current dogma that supports GRP78 expression on the tumor cell surface interfering with the therapeutic(s) and making them as tumor promoters instead. To evaluate the GRP78 localization, we have used ER-/PR-/Her2- (i.e., triple negative) human breast cancer cell line MDA-MB-231 (Caucasian) cultured normally or without serum as well as before or after inducing the ER stress. After establishing the expression of GRP78 mRNA by qPCR and protein by western blotting, we focused on its cell surface expression. Unfixed cells were stained with Concanavalin A (Con A; Specificity = α-D-Mannose, α-D-Glucose, branched mannose), wheat germ agglutinin (WGA; Specificity = (GlcNAc-β-(1,4)-GlcNAc)1-4>β-GlcNAc-NeuAc) as well as with anti-GRP78 antibody followed by their detection by immunofluorescence microscopy using either Rhodamin or Alexa-conjugated secondary antibody. The fluorescence images were captured in a Zeiss microscope with Axiocam camera. Con A and WGA staining provided images of N-glycans on the cell surface and supported the intactness of the cell membrane. On the other hand, GRP78 fluorescence was absent from the surface of these cancer cells. Similar results were also obtained irrespective of cells cultured in the absence of serum and/or in the presence of 1µg/mL of tunicamycin. GRP78 fluorescence however was detected in cells after either fixing them with ice-cold methanol or after permeabilization with digitonin. In addition, western blotting also failed to detect GRP78 in the culture media of MDA-MB-231 cells. We, therefore, conclude that GRP78 is neither expressed on the outer-leaflet of the cell surface of ER-/PR-/Her2- human breast cancer cells MDA-MB-231 nor secreted in the culture media. Supported in part by Susan G. Komen for the Cure BCTR0600582 and NIH/NIMHD G12MD007583 (KB). Citation Format: Jesus E. Serrano, Eva C. Romero-Nutz, Neysharie Sanchez, Aditi Banerjee, Krishna Baksi, Dipak K. Banerjee. GRP78 is neither expressed on ER-/PR-/Her2- human breast cancer cell surface nor secreted in the culture media [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 4509. doi:10.1158/1538-7445.AM2017-4509