Shailesh Singh

CXCL12–CXCR4 interactions modulate prostate cancer cell migration, metalloproteinase expression and invasion

The mechanisms responsible for prostate cancer metastasis are incompletely understood at both the cellular and molecular levels. In this regard, chemokines are a family of small, cytokine-like proteins that induce motility of neoplastic cells, leukocytes and cancer cells. The current study evaluates the molecular mechanisms of CXCL12 and CXCR4 in prostate cancer cell migration and invasion. We report that functional CXCR4 is significantly expressed by prostate cancer cell lines, LNCaP and PC3, when compared with normal prostatic epithelial cells (PrEC). As measured using motility and invasion chamber assays, prostate cancer cells migrated and invaded through extracellular matrix components in response to CXCL12, at rates that corresponded to CXCR4 expression. Anti-CXCR4 antibodies (Abs) significantly impaired the migration and invasive potential of PC3 and LNCaP cells. CXCL12 induction also enhanced collagenase-1 (metalloproteinase-1 (MMP-1)) expression by LNCaP and PC3 cells. Collagenase-3 (MMP-13) was expressed by prostate cancer cells, but it was not expressed by PrEC cells or modulated by CXCL12. CXCL12 increased MMP-2 expression by LNCaP and PC3; however, MMP-9 expression was elevated only in PC3 cells after CXCL12–CXCR4 ligation. PC3 cells also expressed high levels of stromelysin-1 (MMP-3) after CXCL12 stimulation. CXCL12 also significantly increased stromelysin-2 (MMP-10) expression by LNCaP cells. Stromelysin-3 (MMP-11) was expressed by LNCaP cells, but not by PC3 or PrEC cells and CXCL12 induced PC3 MMP-11 expression. Membrane type-1 MMP (MMP-14) was not expressed by PrEC or LNCaP cells, but CXCL12 significantly enhanced MMP-14 expression by PC3 cells. These studies reveal important cellular and molecular mechanisms of CXCR4/CXCL12-mediated prostate cancer cell migration and invasion.

Inhibition of cyclooxygenase-2 decreases breast cancer cell motility, invasion and matrix metalloproteinase expression

BackgroundCyclooxygenase (COX) is the rate-limiting enzyme that catalyzes the formation of prostaglandins. The inducible isoform of COX (COX-2) is highly expressed in aggressive metastatic breast cancers and may play a critical role in cancer progression (i.e. growth and metastasis). However, the exact mechanism(s) for COX-2-enhanced metastasis has yet to be clearly defined. It is well established that one of the direct results of COX-2 action is increased prostaglandin production, especially prostaglandin E2 (PGE2). Here, we correlate the inhibition of COX-2 activity with decreased breast cancer cell proliferation, migration, invasion and matrix metalloproteinase (MMP) expression.MethodsBreast cancer cells (Hs578T, MDA-MB-231 and MCF-7) were treated with selective COX-2 inhibitors (NS-398 and Niflumic acid, NA). Cell proliferation was measured by staining with erythrosin B and counting the viable cells using a hemacytometer. Cell migration and invasion were measured using migration and invasion chamber systems. MMP expression was determined by enzyme immunoassay (secreted protein) and real-time quantitative polymerase chain reaction (mRNA).ResultsOur results show that there is a decline in proliferation, migration and invasion by the Hs578T and MDA-MB-231 breast cancer cell lines in the presence of either low concentrations (1 μM or lower) NA or NS-398. We also report that MMP mRNA and protein expression by Hs578T cells is inhibited by NS-398; there was a 50% decrease by 100 μM NS-398. PGE2 completely reversed the inhibitory effect of NS-398 on MMP mRNA expression.ConclusionOur data suggests that COX-2-dependent activity is a necessary component for cellular and molecular mechanisms of breast cancer cell motility and invasion. COX-2 activity also modulates the expression of MMPs, which may be a part of the molecular mechanism by which COX-2 promotes cell invasion and migration. The studies suggest that COX-2 assists in determining and defining the metastatic signaling pathways that promote the breast cancer progression to metastasis.

Inhibition of IFN-γ-Inducible Protein-10 Abrogates Colitis in IL-10−/− Mice

A deficiency in understanding the steps responsible for colitis is the lack of comprehension for the role chemokines play in mucosal inflammation. IFN-γ-inducible protein-10 (IP-10) and CXCR3 are highly expressed at sites of colitis. Our findings show that IP-10 significantly contributes to the development of Th1 and inflammatory responses. Specifically, IP-10 inhibition in IL-10−/− mice attenuates the associated increases in serum and/or local amyloid A, IL-2, IL-6, TNF-α, IFN-γ, IL-1α, and IL-1β with colitis as compared with IL-10−/− mice that develop colitis similar to human Crohn’s disease. Correspondingly, the rate or intensity of inflammation in IL-10−/− mice treated with anti-IP-10 Abs showed improved scoring of inflammation, compared with control IL-10−/− mice. This study provides important and novel information regarding IP-10 as a target for the treatment of colitis.

Expression and Functional Role of CCR9 in Prostate Cancer Cell Migration and Invasion

Purpose: Metastasis is responsible for most cancer-related deaths; hence, therapies designed to minimize metastasis are greatly needed. The precise cellular and molecular mechanisms used by cancer cells for metastasis are not fully understood; however, the metastatic spread of neoplastic cells is probably related to the ability of these cells to migrate, invade, home, and survive locally. The migration of tumor cells shares many similarities with leukocyte trafficking, which is regulated by chemokine receptor–ligand interactions. The current study evaluates the molecular mechanisms of CCL25 and CCR9 in prostate cancer cell migration and invasion. Experimental Design: In the current study, real-time quantitative polymerase chain reaction, flow cytometry analysis, and in vitro migration as well as invasion chamber analysis (with and without antibody-mediated inhibition) were used to ascertain the biological and functional significance of CCR9 expression by normal prostatic epithelial cells (PrEC) or prostate cancer cell lines (LNCaP-10995 and PC3). Results: We report that functional CCR9 is highly expressed by LNCaP cells and modestly, yet significantly, expressed by PC3 cells when compared with PrEC cells. Neutralization of CCL25–CCR9 interactions impaired the migration and invasion potential of the LNCaP and PC3 cell lines. CCL25 differentially modulated the expression of collagenase-1 or matrix metalloproteinase (MMP)-1, collagenase-3 (MMP-13), stromalysin-2 (MMP-10), stromalysin-3 (MMP-11), and gelatinase-A (MMP-2), but not MMP-3, MMP-7, MMP-8, MMP-9, MMP-12, or MMP-14 in prostate cancer cells. Conclusions: These studies suggest that the expression and activation of CCR9 affect cancer cell migration, invasion, and MMP expression, which together may affect prostate cancer metastasis.

IFN-γ-Inducible Chemokines Enhance Adaptive Immunity and Colitis

T helper type 1 (Th1) cells secreting interferon-γ (IFN-γ) have been closely associated with Crohns disease (CD). Monokine-induced by IFN-γ (MIG), IFN-γ-inducible T cell α chemoattractant (I-TAC), and IFN-γ-inducible protein-10 (IP-10), are chemokines that bind CXCR3 and mediate the chemotaxis of leukocytes. IP-10, MIG, and CXCR3 have been shown to be expressed at sites of CD. The current study stems from our recent findings that IP-10, MIG, and I-TAC significantly contribute to the development of Th1-mediated inflammatory responses. To better understand the role of CXCR3 interactions during CD, we characterized the effects of IP-10, MIG, I-TAC, and CXCR3+ T cells on mucosal immune responses. IP-10, MIG, and I-TAC significantly enhanced antigen-specific serum and mucosal antibodies through Th1-mediated events and CD28 modulation. Additionally, the adoptive transfer of naive CXCR3+ T cells and CD4+CD45RBHI to T cell receptor β (TCRβ) × δ-/- mice resulted in the onset of murine colitis. Taken together, the...

Serum CXCL13 positively correlates with prostatic disease, prostate-specific antigen and mediates prostate cancer cell invasion, integrin clustering and cell adhesion

Chemokines and their corresponding receptor interactions have been shown to be involved in prostate cancer (PCa) progression and organ-specific metastasis. We have recently shown that PCa cell lines and primary prostate tumors express CXCR5, which correlates with PCa grade. In this study, we present the first evidence that CXCL13, the only ligand for CXCR5, and IL-6 were significantly elevated in PCa patient serum compared to serum from subjects with benign prostatic hyperplasia (BPH), or high-grade prostatic intraepithelial neoplasia (HGPIN) as well as normal healthy donors (NHD). Serum CXCL13 levels significantly (p<0.0001) correlated with serum prostate-specific antigen (PSA), whereas serum IL-6 levels significantly (p<0.0003) correlated with CXCL13 serum levels. CXCL13 was found to be a better predictor of PCa than PSA. CXCL13 was highly expressed by human bone marrow endothelial (HBME) cells and osteoblasts (OBs), but not osteoclasts (OCs), following treatment with physiologically relevant levels of interleukin-6 (IL-6). We further demonstrate that CXCL13, produced by IL-6-treated HBME cells, was able to induce PCa cell invasion in a CXCR5-dependent manner. CXCL13-mediated PCa cell adhesion to HBME cells and alpha(v)beta(3)-integrin clustering was abrogated by CXCR5 blockade. These results demonstrate that the CXCL13-CXCR5 axis is significantly associated with PCa progression.

Carcinoma of the gallbladder: role of sonography in diagnosis and staging.

In an attempt to define the sonographic characteristics of gallbladder cancer, we retrospectively analyzed the sonographic findings in 203 cases of gallbladder cancer confirmed by cytology or histopathology.

CCL25 mediates migration, invasion and matrix metalloproteinase expression by breast cancer cells in a CCR9-dependent fashion

Breast cancer (BrCa) is one of the most frequently diagnosed cancers and the second leading cause of cancer-related deaths in North American women. Most deaths are caused by metastasis, and BrCa is characterized by a distinct metastatic pattern involving lymph nodes, bone marrow, lung, liver and brain. Migration of metastatic cells share many similarities with leukocyte trafficking, which are regulated by chemokines and their receptors. The current study evaluates the expression and functional role of CCR9, and its only known ligand, CCL25, in BrCa cell migration and invasion. Quantitative immunohistochemical analysis showed that both moderately and poorly differentiated BrCa tissue expressed significantly more (P<0.0001) CCR9 compared to non-neoplastic breast tissue. Interestingly, poorly differentiated BrCa tissue expressed significantly more (P<0.0001) CCR9 compared to moderately differentiated BrCa tissue. Similarly, CCR9 was highly expressed by the aggressive breast cancer cell line (MDA-MD-231) compared to the less aggressive MCF-7. Migration as well as invasion assays were used to evaluate the functional interaction between CCR9 and CCL25 in BrCa cell lines (MDA-MB-231 and MCF-7). Neutralizing CCR9-CCL25 interactions significantly impaired the migration and invasion of BrCa cells. Furthermore, CCL25 enhanced the expression of MMP-1, -9, -11 and -13 active proteins by BrCa cells in a CCR9-dependent fashion. These studies show CCR9 is functionally and significantly expressed by BrCa (poorly > moderately differentiated) tissue and cells as well as that CCL25 activation of this receptor promotes breast tumor cell migration, invasion and MMP expression, which are key components of BrCa metastasis.

Molecular Basis for the Potency of IL-10-Deficient Dendritic Cells as a Highly Efficient APC System for Activating Th1 Response

Identification and targeting of novel immunobiological factors that regulate the induction of Th1 cells are crucial for designing effective vaccines against certain intracellular pathogens, including Chlamydia. IL-10-deficient dendritic cells (DC) are potent APCs and effective cellular vaccines that activate a high frequency of specific Th1 cells. To elucidate the molecular basis for the potency of the IL-10-deficient APC system, we tested the hypothesis that Chlamydia Ag-primed IL-10 knockout (IL-10KO) DC are quantitatively and qualitatively distinct in their metabolic characteristics relating to T cell activation. Using a combination of RT-PCR, two-dimensional gel electrophoresis, and MALDI-TOF-based proteomics analyses, the transcriptional and translational activities of Chlamydia-pulsed DC from wild-type and IL-10KO mice were assessed. IL-10 deficiency caused early maturation and activation of pulsed DC (i.e., high CD11c, CD40, CD80, CD83, CD86, IL-1, IL-12, and the T cell-attracting chemokine CCL27/CTACK) and consequently an enhanced ability to process and present Ags for a rapid and robust T cell activation. Supporting comparative proteomics revealed further that IL-10 deficient DC possess specific immunobiological properties, e.g., the T cell-attracting chemokine CCL27/CTACK, calcium-dependent protein kinase, and the IL-1/IL-12 inducer, NKR-P1A (CD161), which differentiated them immunologically from wild-type DC that express molecules relating to anti-inflammatory, differentiative, and metabolic processes, e.g., the anti-IL-12 molecule peroxisome proliferator-activated receptor-α and thymidine kinase. Collectively, these results provide a molecular basis for the high Th1-activating capacity of IL-10KO APC and may provide unique immunomodulation targets when designing vaccines against pathogens controlled by T cell immunity.

Differential PsaA-, PspA-, PspC-, and PdB-specific immune responses in a mouse model of pneumococcal carriage.

ABSTRACT Larger numbers of pneumococci were detected in the nasal tract compared to the lung, cervical lymph nodes, and spleen 1, 2, 4, 7, 14, and 21 days after nasal challenge with Streptococcus pneumoniae strain EF3030. In this mouse model of pneumococcal carriage, peripheral S. pneumoniae pneumococcal surface adhesin A (PsaA)-specific humoral responses (immunoglobulin G2a [IgG2a] ≫ IgG1 = IgG2b > IgG3) were significantly higher than pneumococcal surface protein A (PspA)-specific, genetic toxoid derivative of pneumolysin (PdB)-specific, or pneumococcal surface protein C (PspC)-specific serum antibody levels. However, PspA-specific mucosal IgA antibody levels were significantly higher than those against PsaA, PdB, and PspC. In general, both PsaA- and PspA-specific lung-, cervical lymph node-, nasal tract-, and spleen-derived CD4+ T-cell cytokine (interleukin-4, interleukin-6, granulocyte-macrophage colony-stimulating factor, gamma interferon, and tumor necrosis factor alpha) and proliferative responses were higher than those for either PspC or PdB. Taken together, these findings suggest that PsaA- and PspA-specific mucosal responses as well as systemic humoral and T helper cell cytokine responses are predominantly yet differentially induced during pneumococcal carriage.