Inability of Serotonin to Activate the c-Jun N-terminal Kinase and p38 Kinase Pathways in Rat Aortic Vascular Smooth Muscle Cells

BACKGROUND: Serotonin (5-HT, 5-hydroxytryptamine) activates the Extracellular Signal-Regulated Kinase (ERK)/ Mitogen-Activated Protein Kinase (MAPK) pathways, in vascular smooth muscle cells. Parallel MAPK pathways, the c-Jun N-terminal Kinase (JNK) and p38 pathway, are activated by stimulators of the ERK/MAPK pathway. We hypothesized that 5-HT would activate the JNK and p38 pathways in rat vascular smooth muscle cells. RESULTS: Results were determined using standard Western analysis and phosphospecific JNK and p38 antibodies. No significant activation by 5-HT (10(-9) – 10(-5) M; 30 min) of the JNK or p38 pathways, as measured by protein phosphorylation, was observed in any of these experiments. These experiments were repeated in the presence of the serine/threonine phosphatase inhibitor okadaic acid (1 uM) and the tyrosine phosphatase inhibitor sodium orthovanadate (1 uM) to maximize any observable signal. Even under these optimized conditions, no activation of the JNK or p38 pathways by 5-HT was observed. Time course experiments (5-HT 10(-5) M; 5 min, 15 min, 30 min and 60 min) showed no significant activation of JNK after incubation with 5-HT at any time point. However, we detected strong activation of JNK p54 and p46 (5- and 7 fold increases in bands p54 and p46, respectively over control levels) by anisomycin (500 ng/ml, 30 min). Similarly, a JNK activity assay failed to reveal activation of JNK by 5-HT, in contrast to the strong stimulation by anisomycin. CONCLUSION: Collectively, these data support the conclusion that 5-HT does not activate the JNK or p38 pathways in rat vascular smooth muscle cells

A paradigm for the treatment of prostate cancer bone metastases based on an understanding of tumor cell–microenvironment interactions

The pliability of cancer cells to mutate into several different phenotypes in an attempt to find one that will survive and colonize at the metastatic site is a tremendous “hurdle” to overcome in designing novel cancer therapeutics. New targets of therapy are essential if we are to effectively overcome the evasiveness of cancer. The interaction between the tumor cell and the surrounding microenvironment creates a vicious cycle that perpetuates disease survival and progression. The future of cancer therapy resides in the ability to focus on the recruited and exploited relationships of the cancer cell with the host environment. These therapies target cancer cell growth early and interrupt the vicious cycle that is created by the tumor cells interacting with bone components by inhibiting osteoclasts, osteoblasts, stromal cells, and endothelial cells. They alter the bone microenvironment, creating a hostile “soil” that prevents the “seed” from developing into bone metastases and represent a potential new platform for the development of prostate cancer therapeutics. © 2005 Wiley-Liss, Inc.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/48763/1/20522_ftp.pd

Targeted next generation sequencing as a reliable diagnostic assay for the detection of somatic mutations in tumours using minimal DNA amounts from formalin fixed paraffin embedded material

Background Targeted Next Generation Sequencing (NGS) offers a way to implement testing of multiple genetic aberrations in diagnostic pathology practice, which is necessary for personalized cancer treatment. However, no standards regarding input material have been defined. This study therefore aimed to determine the effect of the type of input material (e.g. formalin fixed paraffin embedded (FFPE) versus fresh frozen (FF) tissue) on NGS derived results. Moreover, this study aimed to explore a standardized analysis pipeline to support consistent clinical decision-making. Method We used the Ion Torrent PGM sequencing platform in combination with the Ion AmpliSeq Cancer Hotspot Panel v2 to sequence frequently mutated regions in 50 cancer related genes, and validated the NGS detected variants in 250 FFPE samples using standard diagnostic assays. Next, 386 tumour samples were sequenced to explore the effect of input material on variant detection variables. For variant calling, Ion Torrent analysis software was supplemented with additional variant annotation and filtering. Results Both FFPE and FF tissue could be sequenced reliably with a sensitivity of 99.1%. Validation showed a 98.5%concordance between NGS and conventional sequencing techniques, where NGS provided both the advantage of low input DNA concentration and the detectio

In Vivo Evaluation of AT-101 (R-(-)-Gossypol Acetic Acid) in Androgen-Independent Growth of VCaP Prostate Cancer Cells in Combination with Surgical Castration

AbstractPURPOSE: Upregulation of Bcl-2 family members is a well-established mechanism in the development of androgen-independent prostate cancer. Inhibition of the antiapoptotic proteins Bcl-2 and Mcl-1 may delay the transition to androgen-independent growth. EXPERIMENTAL DESIGN: We have established a prostate cancer model with VCaP prostate cancer cells in vivo to study the transition to androgen independence. Here, we investigated the efficacy of AT-101 (R-(—)-gossypol acetic acid; a pan small molecule inhibitor of Bcl-2, BCI-xL, Mcl-1) in combination with surgical castration to delay the onset of androgen-independent growth in vivo. RESULTS: AT-101 (15 mg/kg, per os (p.o.) 5 days/week) in combination with surgical castration delayed the onset of androgen-independent prostate cancer growth in vivo. In addition, we demonstrate the induction of caspase-9- and caspase-3-dependent induction of apoptosis following AT-101 treatment in vitro which was accompanied by an AT-101-induced downregulation of Bcl-2 and Mcl-1 mRNA and protein expression. CONCLUSIONS: We conclude that AT-101 in combination with surgical castration delays the onset of androgen-independent prostate cancer in vivo by disrupting the antiapoptotic activity of BCl-2 upregulation during the transition to androgen independence. Further studies are needed to define the mechanism of action by which AT-101 attenuates the expression of Bcl-2 and Mcl-1 and to characterize the potential for AT-101 in combination with hormone therapy

Inhibition of Decay-Accelerating Factor (CD55) Attenuates Prostate Cancer Growth and Survival In Vivo

Decay-accelerating factor (CD55) is a member of membrane-bound complement-regulatory proteins. CD55 expression correlates with poor survival in patients with colorectal cancer and has been implicated in the survival and tumorigenesis of blood-borne malignancies. Histologic analysis of clinical specimens from patients with advanced prostate cancer revealed an increase in CD55 expression in prostate tumor epithelial cells. CD55 was shown to be functionally active and to inhibit complement-mediated lysis in PC-3 and DU145 cells. The percentage of lysis was correlative with the CD55 expression profile observed in these prostate cancer cell lines. These data suggest that CD55 is an important regulator of prostate cancer cell survival. As a result, we have hypothesized that CD55 expression on prostate cancer cells promotes cell survival and contributes to the metastatic potential of prostate cancer cells. To determine the role of CD55 in prostate cancer tumorigenesis and metastasis, we generated PC-3(Luc) prostate cancer cells with CD55 siRNA-targeted disruption. We found that PC-3(Luc)/CD55 siRNA constructs in SCID mice resulted in a significant attenuation of overall tumor burden. Further investigation into the mechanisms of CD55-mediated tumor cell/microenvironment interaction is necessary to understand the role of CD55 in tumor cell survival and metastatic lesion formation

CCL2 as an Important Mediator of Prostate Cancer Growth In Vivo through the Regulation of Macrophage Infiltration1

The ability of CCL2 to influence prostate cancer tumorigenesis and metastasis may occur through two distinct mechanisms: 1) a direct effect on tumor cell growth and function, and 2) an indirect effect on the tumor microenvironment by the regulation of macrophage mobilization and infiltration into the tumor bed. We have previously demonstrated that CCL2 exerts a direct effect on prostate cancer epithelial cells by the regulation of their growth, invasion, and migration, resulting in enhanced tumorigenesis and metastasis. Here we describe an indirect effect of CCL2 on prostate cancer growth and metastasis by regulating monocyte/macrophage infiltration into the tumor microenvironment and by stimulating a phenotypic change within these immune cells to promote tumor growth (tumor-associated macrophages). VCaP prostate cancer cells were subcutaneously injected in male SCID mice and monitored for tumor volume, CD68+ macrophage infiltration, and microvascular density. Systemic administration of anti-CCL2 neutralizing antibodies (CNTO888 and C1142) significantly retarded tumor growth and attenuated CD68+ macrophage infiltration, which was accompanied by a significant decrease in microvascular density. These data suggest that CCL2 contributes to prostate cancer growth through the regulation of macrophage infiltration and enhanced angiogenesis within the tumor

Detection and Isolation of Circulating Tumor Cells in Urologic Cancers: A Review

The American Cancer Society has estimated that in 2003, there will be approximately 239,600 new cases of urologic cancer diagnosed and 54,600 urologic cancer-related deaths in the United States. To date, the majority of research and therapy design have focused on the microenvironment of the primary tumor site, as well as the microenvironment of the metastatic or secondary (target) tumor site. Little attention has been placed on the interactions of the circulating tumor cells and the microenvironment of the circulation (i.e., the third microenvironment). The purpose of this review is to present the methods for the detection and isolation of circulating tumor cells and to discuss the importance of circulating tumor cells in the biology and treatment of urologic cancers