Fanghong Chen

BCG directly induces cell cycle arrest in human transitional carcinoma cell lines as a consequence of integrin cross-linking

BackgroundCurrent models of the mechanism by which intravesical BCG induces an anti-tumor effect in urothelial carcinoma propose a secondary cellular immune response as principally responsible. Our group has demonstrated that BCG mediated cross-linking of α51 integrin receptors present on the tumor surface elicits a complex biologic response involving AP1 and NF-κB signaling as well as the transactivation of immediate early genes. This study evaluated the direct biologic effect of cross-linking α5β1 integrin on cell cycle progression and apoptosis in two human urothelial carcinoma cell lines.MethodsTwo independent assays (MTT and Colony forming ability) were employed to measure the effect of α5β1 cross-linking (antibody mediated or BCG) on cellular proliferation. Flow cytometry was employed to measure effect of BCG and α5β1 antibody mediated cross-linking on cell cycle progression. Apoptosis was measured using assays for both DNA laddering and Caspase 3 activation.ResultsResults demonstrate that integrin cross-linking by BCG, or antibody mediated crosslinking of α5β1 resulted in a decrease in proliferating cell number. BCG treatment or α5β1 cross-linking increased the percentage of cells in G0/G1, in both 253J and T24 cell lines. Peptide mediated blockade of integrin binding site using RGDS reversed the effect BCG on both proliferation and cell cycle arrest. Apoptosis in response to BCG was not identified by either DNA laddering or Caspase 3 activation.ConclusionThese findings show that BCG exerts a direct cytostatic effect on human urothelial carcinoma cell lines. Cell cycle arrest at the G1/S interface is a mechanism by which BCG inhibits cellular proliferation. This effect is duplicated by antibody mediated cross-linking of α5β1 and likely occurs as a consequence of crosslink-initiated signal transduction to cell cycle regulatory genes.

MB49 murine urothelial carcinoma: molecular and phenotypic comparison to human cell lines as a model of the direct tumor response to bacillus Calmette-Guerin.

PURPOSE The mouse urothelial carcinoma cell line MB49 is widely used as an in vitro and in vivo model of urothelial carcinoma. Little comparative data exist on the molecular and phenotypic responses of this cell line relative to human cell lines. We compared the effect of bacillus Calmette-Guerin on the MB49 cell line relative to responses previously observed in the human urothelial carcinoma lines T24 (ATCC) and 253J. MATERIALS AND METHODS Molecular end points in MB49 cells after bacillus Calmette-Guerin exposure were signaling pathway activation (NF-kappaB, AP1 and C/EBP), gene expression (IL-6 and p21), HMGB1 release/responsiveness and gene expression profiling at 6 hours. Phenotypic response end points were direct cytotoxicity using dye exclusion, viability on MTT assay, apoptotic sensitivity and cell cycle compartmentalization. RESULTS NF-kappaB, AP1, C/EBP, IL-6 and p21 reporter constructs were activated in MB49 cells in response to bacillus Calmette-Guerin. Gene expression profiles showed an inflammatory/immune clustering response. Bacillus Calmette-Guerin decreased cell viability and induced G1 cell cycle arrest. Treatment of MB49 cells with bacillus Calmette-Guerin induced caspase independent cell death while simultaneously decreasing sensitivity to pro-apoptotic agents. Cell death was associated with release of the necrotic cell death marker HMGB1. MB49 cells expressed HMGB1 receptors and activated intracellular NF-kappaB signaling pathways in response to bacillus Calmette-Guerin. CONCLUSIONS MB49 cells show molecular and phenotypic responses to bacillus Calmette-Guerin that replicate those observed in human urothelial carcinoma lines. MB49 cells appear to be an excellent model in which to study bacillus Calmette-Guerin as an antitumor agent for urothelial carcinoma.

Bacille-Calmette Guèrin induces caspase-independent cell death in urothelial carcinoma cells together with release of the necrosis-associated chemokine high molecular group box protein 1.

To evaluate the ability of bacille‐Calmette Guèrin (BCG) to induce caspase‐independent cell death and release the necrosis‐associated chemokine high molecular group box protein 1 (HMGB1) from urothelial carcinoma (UC) cells; a correlative clinical trial determined if BCG treatment resulted in increased urinary levels of HMGB1.

p21 Expression by human urothelial carcinoma cells modulates the phenotypic response to BCG

PURPOSE The direct phenotypic effects of BCG on human urothelial carcinoma (UC) cells include cell cycle arrest, apoptotic resistance, and caspase-independent cell death. These effects are associated with increased expression of the cyclin dependant kinase inhibitor (CDKI) p21. This study assessed the role of p21 expression in mediating the phenotypic effects observed in response to BCG. MATERIALS AND METHODS Inducible systems for the autocrine expression of p21, or the blockade of p21 expression in response to BCG, were established in the human UC line T24. The effect of increasing or inhibiting p21 expression on tumor phenotype was assessed using assays for cell cycle compartmentalization (flow cytometry), apoptotic sensitivity (caspase 3 activation), and cytotoxicity (vital dye exclusion). RESULTS p21 Overexpression resulted in cell cycle arrest with an increase in the percentage of the cell in G0/G1 phase when compared with the untreated group. p21 Expression decreased basal caspase-3 expression compared with the untreated group, and reversed camptothecin-induced caspase-3 activity compared with camptothecin alone group. p21 Overexpression increased BCGs direct cytotoxicity. shRNA-mediated inhibition of p21 expression in response to BCG failed to reverse BCG-induced changes in cell cycle compartmentalization. p21 Inhibition partially reversed the antiapoptotic effect of BCG. The expression of p21 was required for the direct cytotoxic effect of BCG. CONCLUSIONS p21 Expression is sufficient but not necessary for BCG-induced cell cycle arrest. It is both sufficient and necessary for the full antiapoptotic effect of BCG. p21 Expression alone is not sufficient for caspase-independent cytotoxicity but is necessary for BCGs direct cytotoxic effect.

HMGB1 Release by Urothelial Carcinoma Cells in Response to Bacillus Calmette-Guérin Functions as a Paracrine Factor to Potentiate the Direct Cellular Effects of Bacillus Calmette-Guérin

PURPOSE Prior study demonstrated that HMGB1 release by urothelial carcinoma cells in response to bacillus Calmette-Guérin is required for an in vivo antitumor effect. We evaluated the direct effects of HMGB1 on the in vitro response of urothelial carcinoma cells to bacillus Calmette-Guérin. MATERIALS AND METHODS Two human urothelial carcinoma cell lines were used to study the effect of exogenous HMGB1 alone and combined with bacillus Calmette-Guérin on the tumor cell response to bacillus Calmette-Guérin. Antibody mediated blockade of receptors for HMGB1 or HMGB1 protein was used to determine the contribution of paracrine HMGB1 release to bacillus Calmette-Guérin biological effects. Response end points evaluated included the activation of intracellular signaling pathways, gene transactivation and cytotoxicity. RESULTS Urothelial carcinoma cells expressed the receptor for HMGB1 signaling. Antibody blockade of the RAGE receptor confirmed the dependence of signaling in response to HMGB1 on RAGE function. Exogenous HMGB1 activated cell signaling pathways for NFκB, NRF2 and CEBP. Quantitative reverse transcriptase-polymerase chain reaction on a panel of bacillus Calmette-Guérin responsive genes revealed peak expression resulting from the combination of bacillus Calmette-Guérin and HMGB1. Blockade of paracrine HMGB1 released in response to bacillus Calmette-Guérin using HMGB1 and/or RAGE receptor blocking antibodies showed a significant decrease in gene expression relative to that of bacillus Calmette-Guérin alone. HMGB1 potentiated the cytotoxic effects of bacillus Calmette-Guérin. CONCLUSIONS HMGB1 released by urothelial carcinoma cells after bacillus Calmette-Guérin treatment functions as a paracrine factor to potentiate the urothelial carcinoma cell response to bacillus Calmette-Guérin. This paracrine activity likely contributes to the dependence of an in vivo tumor response on HMGB1 release.

iNOS expression and NO production contribute to the direct effects of BCG on urothelial carcinoma cell biology

OBJECTIVES Evidence suggests that oxidative stress occurring as a consequence of inducible nitric oxide synthase/nitric oxide (iNOS/NO) contributes to the biologic effects of bacille Calmette-Guérin (BCG). Objective of this study is to examine iNOS expression, NO production, and the biologic effect of NO on established intermediate end points for the human urothelial carcinoma cell response to BCG. MATERIALS AND METHODS Quantitative reverse transcriptase-polymerase chain reaction and real-time measurement of NO was used to assess iNOS and NO production, respectively, in 2 human urothelial carcinoma (UC) cell lines, in response to BCG. The effect of blocking NO production using the specific iNOS inhibitor 1400W was determined for multiple intermediate end points characterizing BCGs direct effects on tumor cell biology. Activation of nuclear factor kappa B and nuclear factor (erythroid-derived 2)-like 2 signaling pathways, transactivation of genes, including p21, CD54, IL6, IL8, CXCL1, CXCL3, CCL20, and cytotoxicity, as measured by vital dye exclusion, lactate dehydrogenase release, and 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay were measured in response to BCG with and without iNOS inhibition. RESULTS Exposure of UC cells to BCG significantly increased both iNOS expression and NO production. Inhibition of iNOS activity with 1400W significantly inhibited BCGs direct biologic effect on UC cells for all of the end points evaluated. CONCLUSIONS iNOS expression, NO production, and the associated oxidative stress play a central role in the response of UC cells to BCG exposure. Manipulation of oxidative stress may afford an opportunity to enhance the antitumor effects of BCG.

HMGB1 Release by Urothelial Carcinoma Cells is Required for the In Vivo Antitumor Response to Bacillus Calmette-Guérin

PURPOSE Prior series showed that a portion of urothelial carcinoma cells exposed to bacillus Calmette-Guérin undergoes nonapoptotic cell death and release of the chemokine HMGB1. We evaluated the role of tumor cell derived HMGB1 in mediating the in vivo antitumor effect of bacillus Calmette-Guérin. MATERIALS AND METHODS The murine urothelial carcinoma cell line MB49 was engineered to express a shRNA construct targeting HMGB1. The shRNA expressing cell line underwent characterization to ensure its comparability to the parental MB49 cell line. An orthotopic tumor model was used to compare the in vivo antitumor efficacy of bacillus Calmette-Guérin in the parental cell line (24 control and 24 bacillus Calmette-Guérin treated) vs the HMGB1 knockdown line (23 control and 21 treated). RESULTS Expression of the shRNA construct decreased HMGB1 expression and its release in response to bacillus Calmette-Guérin. The parental and shRNA cell lines showed similar in vitro doubling time and cytotoxicity in response to bacillus Calmette-Guérin. Treatment significantly decreased tumor volume vs controls in parental MB49 tumor bearing mice (p = 0.036). Tumor volume in treated mice inoculated with the shRNA cell line was higher than that in sham treated shRNA controls (p = 0.12). Of the bacillus Calmette-Guérin treated mice tumor volume was significantly lower in parental tumor bearing mice vs the shRNA group (p <0.00001). ANOVA revealed a significant interaction between the cell line (shRNA vs parental) and the bacillus Calmette-Guérin effect (p = 0.0076). CONCLUSIONS The direct tumor response to bacillus Calmette-Guérin, culminating in HMGB1 release, may be an important contributor to the clinical efficacy of bacillus Calmette-Guérin.

Loss of Bacillus Calmette-Guérin Viability Adversely Affects the Direct Response of Urothelial Carcinoma Cells to Bacillus Calmette-Guérin Exposure

PURPOSE The attenuated mycobacterium bacillus Calmette-Guérin is widely used as intravesical immunotherapy for nonmuscle invasive urothelial carcinoma. Previous studies demonstrated that in the laboratory and clinical settings bacillus Calmette-Guérin viability is a variable that correlates with antitumor efficacy. We evaluated how loss of viability impacted a number of molecular and phenotypic intermediate end points that characterize and/or contribute to the direct effect of bacillus Calmette-Guérin on urothelial carcinoma cells. MATERIALS AND METHODS We studied the effect of loss of bacillus Calmette-Guérin viability on the tumor cell response to the treatment in 2 human urothelial carcinoma cell lines. The cellular response to bacillus Calmette-Guérin rendered nonviable by heat killing was compared to the response to viable bacillus. Response end points included the induction of oxidative stress, activation of intracellular signaling pathways, gene transactivation and phenotypic changes. RESULTS Loss of viability resulted in a quantitative decrease in the tumor cell response relative to that of viable bacillus Calmette-Guérin for all measured end points. The decrease in response varied by cell line, ranging from 15% to 100% of the response to viable bacillus. While responses were quantitatively different, nonviable bacillus continued to induce responses that were qualitatively similar to those of bacillus Calmette-Guérin relative to that in untreated controls. CONCLUSIONS Bacillus Calmette-Guérin viability is an important variable influencing the direct tumor cell response to the treatment. Although the magnitude of its effects are attenuated, heat killed bacillus Calmette-Guérin remains active for the induction of bacillus Calmette-Guérin responsive biological end points.

A Synthetic Polyvalent Ligand for α5β1 Integrin Activates Components of the Urothelial Carcinoma Cell Response to Bacillus Calmette-Guérin

PURPOSE Prior study has shown that bacillus Calmette-Guérin binds to and cross-links α5β1 integrins present on the surface of urothelial carcinoma cells. Antibody mediated cross-linking of α5β1 integrins can reproduce signal transduction, gene transactivation and phenotypic changes, similar to those observed in response to bacillus Calmette-Guérin. We evaluated the effect of a synthetic polyvalent ligand for α5β1 on these elements of the tumor response to bacillus Calmette-Guérin. MATERIALS AND METHODS The consensus α5β1 integrin binding tripeptide RGD was linked to a MAP8 backbone to result in an octavalent construct targeting α5β1 integrin. RGD-MAP8 was used to determine its effect on signaling pathway activation (nuclear factor-κB, NRF2 and CEBP), gene expression (p21, interleukin-6 and 8, CXCL1, CXCL2 and CCL20) and cytotoxicity (trypan blue exclusion and HMGB1 release) in human urothelial carcinoma cells. Results were compared to those of treatment with bacillus Calmette-Guérin or the missense peptide GRD-MAP8. RESULTS The RDG-MAP8 construct significantly increased nuclear factor-κB signaling and p21 expression relative to controls. Compared to bacillus Calmette-Guérin treatment, only p21 expression was comparable for cells treated with RGD-MAP8, averaging 70% of bacillus Calmette-Guérin induced expression. RGD-MAP8 failed to have a significant effect on CEBP or NRF2 activation, gene expression or cell viability. CONCLUSIONS Intracellular signaling, gene transactivation and phenotypic changes in response to RGD-MAP8 were qualitatively and quantitatively different than those observed in response to bacillus Calmette-Guérin. Results suggest that while α5β1 integrin cross-linking contributes to the bacillus Calmette-Guérin response, it alone is insufficient to duplicate the full spectrum of bacillus Calmette-Guérin induced changes in urothelial carcinoma cell biology.

The Effects of Physiological Estrogen Concentration on the Immune Response of Urothelial Carcinoma Cells to Bacillus Calmette-Guérin

PURPOSE Meta-analysis has shown that bacillus Calmette-Guérin is less effective in females undergoing treatment for urothelial carcinoma. Urothelial carcinoma cells express immune regulatory proteins as a consequence of bacillus Calmette-Guérin induced, nuclear factor κB signaling. Nuclear factor κB is influenced by estrogen receptor binding. We evaluated the effect of the physiological estradiol concentration on the expression of bacillus Calmette-Guérin induced, nuclear factor κB regulated immune proteins. MATERIALS AND METHODS We determined the estrogen receptor expression status of human urothelial carcinoma cell lines by reverse transcriptase-polymerase chain reaction. The functional status of estrogen receptor signaling was established using estrogen receptor reporter constructs. We used gene expression profiling of urothelial carcinoma cells combined with reverse transcriptase-polymerase chain reaction to identify the nuclear factor κB dependent immune regulatory proteins expressed in response to bacillus Calmette-Guérin. We determined the influence of the estradiol concentration on bacillus Calmette-Guérin dependent interleukin-6 and 8, chemokine (c-x-c motif) ligands 1 and 2, and chemokine (c-c motif) ligand 20 gene expression by quantitative reverse transcriptase-polymerase chain reaction. RESULTS Urothelial carcinoma cell lines expressed functional estrogen receptor. Nuclear factor κB signaling was inhibited by estradiol in a dose and estrogen receptor dependent manner. Urothelial carcinoma cell expression of interleukin-6 and 8, chemokine (c-x-c motif) ligands 1 and 2, and chemokine (c-c motif) ligand 20 was up-regulated in response to bacillus Calmette-Guérin in a nuclear factor κB dependent manner. There was a significant dose dependent effect of estradiol on the expression of these genes in bacillus Calmette-Guérin treated urothelial carcinoma cells. CONCLUSIONS The physiological concentration of estrogen influences nuclear factor κB signaling and bacillus Calmette-Guérin dependent gene expression. Serum estradiol fluctuations in women may influence the response of urothelial carcinoma to intravesical bacillus Calmette-Guérin treatment.