Rakesh K. Singh

IL-8 Directly Enhanced Endothelial Cell Survival, Proliferation, and Matrix Metalloproteinases Production and Regulated Angiogenesis

IL-8, a member of the chemokine family, has been shown to play an important role in tumor growth, angiogenesis, and metastasis. The objective of this study was to determine the mechanism of IL-8-mediated angiogenesis. We examined the direct role of IL-8 in angiogenesis by examining IL-8 receptor expression on endothelial cells and their proliferation, survival, and matrix metalloproteinases (MMPs) production. We demonstrate that HUVEC and human dermal microvascular endothelial cells constitutively express CXCR1 and CXCR2 mRNA and protein. Recombinant human IL-8 induced endothelial cell proliferation and capillary tube organization while neutralization of IL-8 by anti-IL-8 Ab blocks IL-8-mediated capillary tube organization. Incubation of endothelial cells with IL-8 inhibited endothelial cell apoptosis and enhanced antiapoptotic gene expression. Endothelial cells incubated with IL-8 had higher levels of Bcl-xL:Bcl-xS and Bcl-2:Bax ratios. Furthermore, incubation of endothelial cells with IL-8 up-regulated MMP-2 and MMP-9 production and mRNA expression. Our data suggest that IL-8 directly enhanced endothelial cell proliferation, survival, and MMP expression in CXCR1- and CXCR2-expressing endothelial cells and regulated angiogenesis.

Autocrine role of interleukin-8 in induction of endothelial cell proliferation, survival, migration and MMP-2 production and angiogenesis.

Interleukin-8 (IL-8/CXCL8), a paracrine angiogenic factor, modulates multiple biologic functions in CXCR1 and CXCR2 expressing endothelial cells. Several reports suggest that inflammation, infection, cellular stress and tumor presence regulate IL-8 production in endothelial cells. In the present study, we test the hypothesis that IL-8 regulates multiple biological effects in endothelial cells in an autocrine manner. We examined the autocrine role of IL-8 in regulating angiogenesis by using a neutralizing antibody to IL-8, CXCR1 or CXCR2 in human vein umbilical endothelial cell (HUVEC) and human dermal microvascular endothelial cell (HMEC). Neutralizing antibody to IL-8, CXCR1 or CXCR2 inhibited endothelial cell proliferation, and MMP-2 production as compared to cells cultured with medium alone or control antibody. In addition, we observed that the number of apoptotic cells was significantly higher in anti-IL-8, anti-CXCR1 and anti-CXCR2 treated endothelial cells, which coincided with decreased survival-associated gene expression. We observed reduced migration of endothelial cells treated with anti-IL-8 and anti-CXCR2 antibody, but not anti-CXCR1 antibody as compared to controls. Further, we observed an inhibition of capillary tube formation and neovascularization following treatment with anti-IL-8, anti-CXCR1 and anti-CXCR2 antibodies. Together these data suggest that IL-8 functions as an important autocrine growth and angiogenic factor in regulating multiple biological activities in endothelial cells.

Chemokines in tumor angiogenesis and metastasis

Chemokines are a large group of low molecular weight cytokines that are known to selectively attract and activate different cell types. Although the primary function of chemokines is well recognized as leukocyte attractants, recent evidences indicate that they also play a role in number of tumor-related processes, such as growth, angiogenesis and metastasis. Chemokines activate cells through cell surface seven trans-membranes, G-protein-coupled receptors (GPCR). The role played by chemokines and their receptors in tumor pathophysiology is complex as some chemokines favor tumor growth and metastasis, while others may enhance anti-tumor immunity. These diverse functions of chemokines establish them as key mediators between the tumor cells and their microenvironment and play critical role in tumor progression and metastasis. In this review, we present some of the recent advances in chemokine research with special emphasis on its role in tumor angiogenesis and metastasis.

Distinct Expression of CXCL8 and Its Receptors CXCR1 and CXCR2 and Their Association With Vessel Density and Aggressiveness in Malignant Melanoma

We examined the expression of CXCL8 (interleukin-8), its receptors, CXCR1 and CXCR2, and vessel density in human melanoma by immunohistochemical analysis of tumors from different Clark levels, depths, and thicknesses. Expression of CXCL8 and CXCR2 was lower in Clark level I and II specimens than in level III through V specimens and metastases. CXCR1 expression was observed ubiquitously in the majority of human melanoma tumor specimens irrespective of disease state, with the highest intensity in Clark level III specimens. We observed a significant difference in CXCL8 and CXCR2 expression between thin (<or=0.75 mm) and thick (>0.75 mm) melanomas and between thin and metastatic lesions. Positive correlations were observed between Clark level and CXCL8 or CXCR2 and between thickness and CXCR2 expression. We found no correlation between vessel density and Clark level or thickness. Our data suggest that expression of CXCL8 and CXCR2 contributes to aggressive growth and metastasis in human malignant melanoma. Consistent with the transition from radial to vertical growth phase melanoma, expression of CXCL8 and its receptor, CXCR2, may be key in the switch to an aggressive, more metastatic phenotype.

Expression of CXCR1 and CXCR2 receptors in malignant melanoma with different metastatic potential and their role in interleukin-8 (CXCL-8)-mediated modulation of metastatic phenotype

In the present study, we examined the autocrine/paracrine role of IL-8 in melanoma growth and metastasis by analyzing the expression and functional significance of IL-8 receptors, CXCR1 and CXCR2 in human malignant melanoma cells with different metastatic potential. CXCR1 and CXCR2 mRNA and protein levels were analyzed by reverse trannscriptase-based polymerase chain reaction, immunohistochemistry, immunoprecipitation, flow cytometry and ligand binding assay in melanoma cells in vitro and xenografted in nude mice. Melanoma cells constitutively expressed CXCR1 and CXCR2 mRNA and protein. Highly metastatic A375SM cells expressed higher levels of CXCR1 and CXCR2 mRNA and protein in vitro and in vivo as compared to low metastatic A375P and non-metastatic SBC-2 melanoma cells. Treatment of SBC-2 and A375P cells with exogenously added recombinant IL-8 significantly enhanced their proliferation and invasive potential. Further neutralizing antibodies to CXCR1 and CXCR2 inhibited proliferation and invasive potential of unstimulated and IL-8-stimulated A375P cells. In summary, the data suggest that constitutive expression of CXCR1 and CXCR2 play an important role regulating the IL-8-mediated metastatic phenotype in human malignant melanoma cells.

Cysteine cathepsin S as an immunomodulatory target: present and future trends.

Objective: Antigen presentation is the key to immune response. Cathepsin S plays a major role in the degradation of the invariant peptide chain associated with the major histocompatibility complex and thus affects antigen presentation. This review will focus on the recent developments made in field of cysteine cathepsins especially cathepsin S and their future prospects as a therapeutic target. Methods: Selective cathepsin inhibitors for targeting autoimmune disorders, atherosclerosis, osteoporosis, osteoarthritis and cancer are being pursued by many pharmaceutical companies. Recent publications in this field have been used as references to evaluate the current and future trends in cathepsin S inhibitors as an immunomodulatory target. Conclusions: The temporal and spatial position occupied by cathepsin S in immune presentation, gives rise to the hope that an inhibitor would impart selectivity with a lesser propensity for side effects than other immunosuppressive agents.

Constitutive expression of growth regulated oncogene (gro) in human colon carcinoma cells with different metastatic potential and its role in regulating their metastatic phenotype

The purpose of this study was to examine the expression and functional significance of the growth-regulated oncogene (gro) family in human colon carcinoma growth and metastasis. We examined constitutive expression of CXCL1 (gro-α), CXCL2 (gro-β), CXCL3 (gro-γ) and their receptor, CXCR2 in human colon carcinoma cells with different metastatic potentials. Non-metastatic and low metastatic cells expressed lower levels of CXCL1 and CXCR2 mRNA and protein as compared to high metastatic colon carcinoma cells. No difference in CXCL2 and CXCL3 mRNA expression levels was observed. Colon carcinoma cells expressing higher levels of CXCL1 exhibit increased proliferation and invasive potential. Furthermore, exogenous addition of recombinant human CXCL1 significantly enhanced the proliferation and invasiveness of colon carcinoma cells. Furthermore, treatment of KM12C cells with exogenous CXCL1 enhanced their invasiveness. Neutralizing antibody to CXCL1 in combination with antibody to CXCR2 inhibited highly metastatic KM12L4 (high CXCL1 expressor) cell proliferation. These data demonstrate that the constitutive expression of CXCL1 and its receptor CXCR2 is associated with metastatic potential and modulates colon cancer cell proliferation and an invasive phenotype.

Small molecule antagonists for CXCR2 and CXCR1 inhibit human colon cancer liver metastases

CXCR1 and CXCR2 are G-protein coupled receptors, that have been shown to play important role in tumor growth and metastasis, and are prime targets for the development of novel therapeutics. Here, we report that targeting CXCR2 and CXCR1 activity using orally active small molecule antagonist (SCH-527123, SCH-479833) inhibits human colon cancer liver metastasis mediated by decreased neovascularization and enhanced malignant cell apoptosis. There were no differences in primary tumor growth. These studies demonstrate the important role of CXCR2/1 in colon cancer metastasis and that inhibition of CXCR2 and CXCR1, small molecule antagonists provides a novel therapeutic strategy.

Flt3 ligand and granulocyte-macrophage colony-stimulating factor preferentially expand and stimulate different dendritic and T-cell subsets.

OBJECTIVE Mechanisms of T-cell stimulation by Flt3 ligand (Flt3L) and granulocyte-macrophage colony-stimulating factor (GM-CSF) remain unclear. Herein, we compared the effects of Flt3L and GM-CSF on the expansion of dendritic cells (DC) and T-cell subsets and cytokine expression. METHODS Naïve and effector/memory T cells were analyzed by flow cytometry (FC). CD4(+) and CD8(+) T cells and CD11c(+)CD11b(dull/-)(DC1) and CD11c(+)CD11b(+) (DC2) subsets were isolated and the frequency of IFN-gamma-, IL-12- (type 1) and IL-4-, IL-10 (type 2)-producing cells and cytokine mRNA expression evaluated. RESULTS Flt3L expanded both DC1 and DC2 subsets with a significantly higher percentage and number of DC1 than DC2, while GM-CSF preferentially expanded the DC2 subset. Isolated DC1 from Flt3L-injected mice had significantly higher levels of IL-12 (p40) than IL-10, while the converse occurred with DC2. The numbers of naïve and memory T cells were elevated in mice that received Flt3L or GM-CSF. However, the number of memory CD4(+) and CD8(+) T cells was significantly increased in Flt3L as compared to GM-CSF cohorts. While GM-CSF increased the frequency of both type 1 and type 2 cytokine-producing cells, Flt3L significantly augmented the frequency of type 1 T cells. CONCLUSIONS In contrast to GM-CSF, Flt3L preferentially induces the expansion of type 1 T cells. The mechanism of Flt3L-induced T-cell stimulation is associated with the expansion of the IL-12 (p40)-producing DC1 and memory T cells.

Monocytes from mobilized stem cells inhibit T cell function.

Granulocyte‐macrophage colony‐stimulating factor, mobilized peripheral blood stem cell (PSC) products, and peripheral blood leukocytes posttransplantation contain cells that cause allogeneic and autologous T cell apoptosis. Isolation and characterization of these cells demonstrated that they were low‐density (Percoll fractionation) CD14+ monocytes. T cells in PSC products have a depressed phytohemagglutinin (PHA) mitogenic response; however, purified CD4+ or CD8+ T cells exhibit a statistically normal mitogenic function. Furthermore, no T cell inhibitory activity was observed in CD14+, CD4+, and CD8+ cell‐depleted fractions enriched in CD4–CD8–TCRα/β+ T cells. Inhibition of T cell function by CD14+ monocytes required cell‐cell contact, and the analyses of DNA fragmentation by Southern and TUNEL analysis demonstrates an activation‐induced T cell apoptosis in the presence of CD14+ monocytes. Reverse‐transcriptase polymerase chain reaction studies suggested that high levels of interleukin‐10 or tumor necrosis factor gene transcripts in the PSC products may contribute to the inhibition of T cell function. J. Leukoc. Biol. 61: 583–591; 1997.