Blockade of leukocyte haptokinesis and haptotaxis by ketoprofen, diclofenac and SC-560.

BACKGROUND: Nonsteroidal anti-inflammatory drugs (NSAID) represent a one of the most widely used anti-inflammatory substances. Their anti-inflammatory effects are mainly based on inhibition of cyclooxygenase. The potential direct effect of NSAID on leukocyte migration was poorly investigated. Using time-lapse microscopy and 96-well fluorescence-based assay, we studied the effect of three different NSAID, ketoprofen, diclofenac and SC-560, on leukocyte haptokinesis and haptotaxis in vivo and in vitro. RESULTS: NSAID induced an immediate inhibiting effect on leukocyte migration both in vitro and in vivo. This effect was dose-dependent and was not restricted to a specific type of leukocytes. The inhibition of leukocyte migration by NSAID was partially re-stored after removal of inhibiting agent. Only complete blockade of leukocyte migration was accompanied by a strong reduction of [Ca(2+)]i. CONCLUSIONS: NSAID strongly supress leukocyte migration. The results of the present study may have important clinical implications since blockade of leukocyte migration can be achieved after topical application of NSAID.

Rapamycin inhibits proliferation and migration of hepatoma cells in vitro.

BACKGROUND: Systemic immunosuppression represents the major factor for cancer recurrence after orthotopic liver transplantation for hepatocellular carcinoma (HCC). Rapamycin is an immunosuppressant with unique antitumoral properties. Although rapamycin has been successfully used in HCC patients after liver transplantation, the detailed mechanisms of rapamycin action on tumor cells are poorly understood. METHODS: Two HCC cell lines (PLC5 and HuH7) were used to evaluate the effect of rapamycin. Tumor cell proliferation was analyzed using cell counting and BrdU incorporation assay. Expression of phosphorylated Akt was studied using enzyme linked immunosorbent assay. Digital time-lapse microscopy was utilized to measure tumor cell migration in vitro. RESULTS: Rapamycin induced a strongly dose-dependent inhibition of tumor cell proliferation in both HCC cell lines. Additionally, rapamycin inhibited activation of Akt phosphorylation and tumor cell migration after prolonged treatment. CONCLUSIONS: Rapamycin suppresses tumor progression due to inhibition of phosphorylated Akt, cell proliferation, and migration. The data of the present study strengthen clinical implications of rapamycin after liver transplantation with HCC.

Promotion of tumor cell migration by extracellular matrix proteins in human pancreatic cancer.

OBJECTIVES: The pathogenesis of pancreatic carcinoma is driven by the tumor cells ability to migrate causing invasion and metastases. The correlation between the aberrant expression of basement membrane proteins and the process of tumor invasion and metastasis has not been fully determined. METHODS: In the present study, the influence of laminin, fibronectin, and collagen type IV on migratory activity of 5 different cell lines has been investigated at the level of a single tumor cell using 3-dimensional time-lapse microscopy. RESULTS: All investigated cell lines have shown a high baseline migration that varied between 6.2 +/- 3.6 and 20.6 +/- 6.8 microm/h. The addition of laminin, fibronectin, and collagen type IV to collagen type I matrix has significantly increased tumor cell migration. Tumor cell migration was strongly inhibited after treating the tumor cells with anti-beta1 monoclonal antibodies. An abundant and continuous expression of laminin, fibronectin, and collagen type IV was found on the basement membrane of perineurium, which sharply promoted tumor cell invasion. CONCLUSIONS: The continuous presentation of the basement membrane proteins by perineurium contributes to the affinity of pancreatic cancer cells for the perineural tumor invasion. Blockade of integrins could represent a possible approach to control the basement membrane-guided tumor spread.

Consensus transcriptome signature of perineural invasion in pancreatic carcinoma.

Perineural invasion, the growth of tumor cells along nerves, is a key feature of pancreatic cancer. The cardinal symptom of pancreatic cancer, abdominal pain often radiating to the back, as well as the high frequency of local tumor recurrence following resection are both attributed to the unique ability of pancreatic tumor cells to invade the neuronal system. The molecular mechanisms underlying the neuroaffinity of pancreatic tumors are not completely understood. In this study, we developed a novel method to monitor ex vivo perineural invasion into surgically resected rat vagal nerves by different human pancreatic tumor cell lines. Genome-wide transcriptional analyses were employed to identify the consensus set of genes differentially regulated in all highly nerve-invasive (nerve invasion passage 3) versus less invasive (nerve invasion passage 0) pancreatic tumor cells. The critical involvement of kinesin family member 14 (KIF14) and Rho-GDP dissociation inhibitor beta (ARHGDIbeta) in perineural invasion was confirmed on RNA and protein levels in human pancreatic tumor specimens. We found significant up-regulation of KIF14 and ARHGDIbeta mRNA levels in patients with pancreatic cancer, and both proteins were differentially expressed in tumor cells invading the perineural niche of pancreatic cancer patients as detected by immunohistochemistry. Moreover, functional knockdown of KIF14 and ARHGDIbeta using small interfering RNA resulted in altered basal and/or perineural invasion of pancreatic tumor cells. Our work provides novel insights into the molecular determinants of perineural invasion in pancreatic cancer. The established nerve invasion model and the consensus signature of perineural invasion could be instrumental in the identification of novel therapeutic targets of pancreatic cancer as exemplified by KIF14 and ARHGDIbeta.

Active leukocyte crawling in microvessels assessed by digital time-lapse intravital microscopy.

OBJECTIVE: The ability of active movement is an important feature of leukocytes. Here, we used a hybrid technique that combines intravital microscopy and digital time-lapse video microscopy to investigate the physiology and molecular mechanisms of intravascular leukocyte movement. METHODS: Intravital microscopy of mesenteric venules was performed in male, Wistar rats using digital video recording and time-lapse image compression. The leukocyte movement and extravasation were analyzed after local application of TNF-alpha, after blockade of endothelial (anti-ICAM-1 antibody) and leukocyte (anti-CD18 antibody) adhesion molecules. Additionally, the migratory activity of isolated leukocytes in collagen gel was analyzed and compared with their intravascular locomotion. RESULTS: Adherent leukocytes showed an active intraluminal crawling along the endothelial lining. Most permanent stickers (84 +/- 13%) crawled actively on the intraluminal site of venules. Baseline measurement of leukocyte crawling velocity yielded an average 9.0 +/- 1.8 mum/min that was not significantly different from crawling velocity of extravascular leukocytes (8.9 +/- 4.5 mum/min). The maximum distance of leukocyte crawling observed was 150 microm. The maximum time of crawling was 15 min. Intraluminal crawlers traveled over a mean distance of 35 +/- 17 mum with the average duration of 5.4 +/- 1.4 min. Under unstimulated conditions, almost all crawling leukocytes detached from the endothelium and did not migrate through the vascular wall. TNF-alpha induced a significant increase of leukocyte extravasation. Anti-ICAM-1 and anti-CD18 antibodies significantly reduced leukocyte crawling. The proportion of isolated migrating leukocytes in collagen gel (87% +/- 6%) was not significantly different from the percentage of intravascular crawling leukocytes in vivo. CONCLUSIONS: The method of digital time-lapse intravital microscopy represents an advantageous technology for the investigation of intravascular, transendothelial, and extravascular migration of leukocytes. Using this technology, we showed that leukocyte-endothelial-interactions are an active and dynamic process. This process involves long-time (several minutes) crawling of leukocytes along the endothelium and, finally, detachment from the endothelium. Intravascular leukocyte crawling reflects the migratory potential of circulating leukocytes and strongly depends on the expression of adhesion molecules. For extravasation, an additional pro-inflammatory stimulus is required.