Thrombin enhances adhesion in pancreatic cancer in vitro through the activation of the thrombin receptor PAR 1.

BACKGROUND: Thrombin, the central enzyme of the coagulation cascade, induces proliferation in different solid tumours. The effect is mainly mediated through the functional thrombin receptor PAR 1, a member of the G-protein coupled receptor family. The aim of this study was to assess the role of thrombin on adhesion of pancreatic cancer to extracellular matrix proteins and endothelial cells in vitro. MATERIALS AND METHODS: The human pancreatic adenocarcinoma cell line MIA PaCa-2 was treated with thrombin and the thrombin-receptor-activating peptide (TRAP), respectively. As a control the cells were pre-incubated with the thrombin-receptor-inhibiting peptide (T1). The cells were incubated on microtiter plates, which were pre-coated with extracellular matrix proteins (fibronectin, laminin, collagen IV) or human umbilical vein endothelial cells (HUVECs), for 30 and 60 min, respectively. The number of adherent cells were measured using the MTT method. ANOVA was used for statistical analysis. RESULTS: Thrombin enhanced the adhesion of MIA PaCa-2 cells to extra-cellular matrix proteins and endothelial cells significantly (P< or =0.001). The effects of thrombin could be mimicked by TRAP. Pre-incubation with T1 inhibited the effect. CONCLUSION: Thrombin enhances adhesion of pancreatic adenocarcinoma to extracellular matrix proteins and endothelial cells in vitro. The effect is mediated through the thrombin receptor PAR 1. The results emphasize the role of thrombin and PAR 1 in pancreatic tumour biology.

Detection of tumor cells in peritoneal lavages from patients with gastrointestinal cancer by multiplex reverse transcriptase PCR.

BACKGROUND/AIMS: Cytological examination of peritoneal lavages is a useful predictor of peritoneal recurrence in gastrointestinal carcinoma patients. Nevertheless, it may be inadequate for those patients with lavages containing only few cancer cells. In the present study, sensitive detection of free cancer cells could be achieved through amplification of cytokeratin 19, carcinoembryonic antigen, alpha-fetoprotein mRNAs by means of multiplex reverse transcriptase polymerase chain reaction and nested polymerase chain reaction. METHODOLOGY: The multiplex reverse transcriptase polymerase chain reaction assay was used to examine lavage samples from 64 patients with various gastrointestinal malignant lesions (colorectal n = 27; duodenal carcinoma n = 1; gastric n = 7; pancreatic n = 4; hepatocellular carcinoma n = 2; gallbladder n = 1; cholangiocellular carcinoma n = 2 and 20 colorectal liver metastases. Specificity was assessed by examination of 15 donors without malignancies. In addition, nested polymerase chain reaction was used to improve the sensitivity of the assay for the detection of alpha-fetoprotein transcripts. RESULTS: Peritoneal lavages from 12 of 64 gastrointestinal carcinoma patients were positive for carcinoembryonic antigen mRNA. Carcinoembryonic antigen proved a specific marker, as no false-positives were detected in any patients without gastrointestinal cancer. alpha-fetoprotein mRNA was detected exclusively in peritoneal lavages from tumor patients, i.e., in 16 of 27 colon cancer patients, 14 of 20 patients with colorectal liver metastasis, 2 of 7 patients with gastric cancer, two patients with hepatocellular carcinoma and 2 of 4 patients with pancreatic cancer. Cytokeratin 19 mRNA was not found a useful marker, since control patients without malignancies were also positive. CONCLUSIONS: Our data suggest that carcinoembryonic antigen- and alpha-fetoprotein mRNA in peritoneal lavage are potentially useful specific markers for early diagnosis of metastasis of gastrointestinal cancer. It has been shown that alpha-fetoprotein-specific nested reverse transcriptase polymerase chain reaction can detect not only hepatocellular carcinoma cells, but also malignant cells from other gastrointestinal carcinomas. In contrast, cytokeratin 19 mRNA lacks specificity for gastrointestinal cancer.

Reduction of intrafollicular apoptosis in chemotherapy-induced alopecia by topical calcitriol-analogs.

Chemotherapy-induced alopecia is thought to result from cytotoxic and apoptosis-related damage to the hair follicle. This study was designed to confirm whether keratinocyte apoptosis is indeed induced in growing (= anagen) hair follicles of C57 BL/6 mice after the injection of cyclophosphamide, using improved methods for histologic detection of apoptotic cells in murine skin. More importantly, we asked whether topical calcitriol-analogs are able to modulate cyclophosphamide-induced apoptosis in vivo, because there are conflicting reports on the effects of calcitriols on apoptosis in vitro. Anagen was induced in telogen mice on day 0 by depilation. Starting on day 5 post-depilation, the back skin of mice was topically treated with either 0.2 microg 1,25-dihydroxyvitamin D3, 2.0 microg calcipotriol, 0.02 microg KH 1060, or vehicle (ethanol) only. On the last day of treatment (i.e., day 9 post-depilation), all mice received 150 mg cyclophosphamide i.p. per kg as a single dose to induce alopecia, or vehicle (aqua dist.). Analysis of the treated skin by in situ-end labeling (using a modified terminal UTP nucleotide end labeling technique suitable for murine skin), by Hoechst 33342 stain, and by DNA electrophoresis on days 10 and 14, revealed the induction of massive apoptosis in cyclophosphamide-treated anagen hair bulbs, which was most prominent on day 10, whereas controls showed no follicular apoptosis. The calcitriol-pretreated groups demonstrated a significant reduction of apoptosis, with a maximal inhibition seen on day 14. This confirms that cyclophosphamide indeed induces massive keratinocyte apoptosis in anagen hair follicles, and provides evidence that topical calcitriol-analogs can suppress epithelial cell apoptosis in vivo. The mouse model employed here offers an excellent tool for dissecting the as yet poorly understood controls of keratinocyte apoptosis in situ and its pharmacologic manipulation.

Control of hair growth with parathyroid hormone (7-34).

Parathyroid hormone (PTH) related peptide (PTHrP) is thought to influence the proliferation and differentiation of the epidermis and hair follicle. As a means of elucidating the biologic function of PTHrP on the hair follicle, a PTHrP analog PTH (7-34), which is a PTH/PTHrP receptor antagonist, was given intraperitoneally twice daily to C57 BL/6 mice at different stages of the hair cycle. PTH (7-34) induced 99 +/- 4.5% (mean +/- SEM) of resting telogen hair follicles into a proliferative (anagen) state, whereas 100% of the hair follicles in the control group remained in telogen. To determine whether this peptide influenced the progression of the hair follicles from anagen to catagen (hair follicle maturation and regression), groups of mice that were either spontaneously in or induced to anagen received either PTH (7-34) or placebo. Morphometric analysis of the hair follicles from the middle back region of the spontaneous anagen mice that received PTH (7-34) revealed that 19 +/- 4% (mean +/- SEM) of the follicles were in anagen VI, whereas none (0%) were in anagen in the control group. Similarly, in induced anagen mice treated with PTH (7-34), 22.3 +/- 1.4 (mean +/- SEM) of the follicles were in anagen VI compared to only 1.3 +/- 0.7% in the control mice. Together these observations suggest that PTHrP is a hair follicle morphogen that may be a major factor responsible for controlling the hair cycle. These studies provide a new insight for development of PTHrP analogs for a wide variety of disorders related to disturbances of hair cycling.

Preferential histiotypic expression of CD44-isoforms in human lung cancer.

The CD44 transmembrane glycoprotein is expressed in most adult tissues and in the majority of neoplasias. Due to alternative splicing, this cell adhesion molecule exists in multiple isoforms some of which have been associated with specific types of tumours as well as with increased tumour metastasis. In this study, we have looked at the level and type of CD44 expression in lung cancer which represents a histologically heterogenous form of cancer composed of small cell lung carcinoma (SCLC) and non-small cell lung carcinoma (NSCLC), the latter subgroup comprising adenocarcinoma (ADC), bronchio-alveolar carcinoma (BAC), large cell carcinoma (LCC), and squamous cell carcinoma (SCC). We analysed 20 lung cancer cell lines and 64 primary tumours by RT-PCR and immunohistochemical detection of the CD44 standard and variant protein isoforms. Our results suggest that (i) CD44 is expressed in all histologically distinct subsets of lung cancer with a tendency SCC > BAC > ADC > LCC > SCLC, (ii) expression of the CD44 isoforms v5, v7, v8, and, most notably that of CD44 exon v6, strongly correlates with tumours of squamous cell and bronchio-alveolar carcinoma origin, tumours which commonly exhibit a comparatively low metastasizing potential, and (iii) the expression of CD44 isoforms is independent from the tumour size and lymph node status at surgery, the proliferative status of the tumour cell population (Ki67 antigen expression) and the histopathological grading (G1 to G3). Only non-differentiated tumours (G4), which were restricted to SCLC and LCC samples revealed markedly reduced CD44 standard and isoform antigen. In conclusion, our data point to a clear histiotype-related pattern of CD44 variant expression preferentially that of CD44v6 in SCC and BAC.

Topical calcitriol enhances normal hair regrowth but does not prevent chemotherapy-induced alopecia in mice.

Using a murine model that mimics chemotherapy-induced alopecia (CIA) in humans particularly well, we show here that in contrast to previously reported CIA-protective effects in neonatal rats, topical calcitriol does not prevent CIA in adolescent mice but enhances the regrowth of normally pigmented hair shafts. When, prior to injecting 1 X 120 mg/kg cyclophosphamide i.p., 0.2 microg calcitriol or vehicle alone were administered topically to the back skin of C57BL/6 mice with all hair follicles in anagen, no significant macroscopic differences in the onset and severity of CIA were seen. However, hair shaft regrowth after CIA, which is often retarded and patchy, thus displaying severe and sometimes persistent pigmentation disorders, was significantly accelerated, enhanced, and qualitatively improved in test compared with control mice. Histomorphometric analysis suggests that this is related to the fact that calcitriol-pretreated follicles favor the "dystrophic catagen pathway" of response to chemical injury, ie., a follicular repair strategy allowing for the unusually fast reconstruction of a new, undamaged anagen hair bulb. Thus, it may be unrealistic to expect that topical calcitriol can prevent human CIA, but topical calcitriols may well enhance the regrowth of a normal hair coat.

Expression of bcl-2--protein in small cell lung cancer.

The expression of the proto-oncogene bcl-2, whose main function appears to be an inhibition of apoptosis, was investigated in 164 cases of primary small cell lung cancer by means of immunohistochemistry in a retrospective analysis. One-hundred twenty-five cases (76%) demonstrated expression of bcl-2. There was no difference in serum LDH levels and proliferative activity between the two groups. An analysis revealed a median survival time of 12 months for patients with bcl-2 positive tumors compared to 9.5 months for patients with bcl-2 negative tumors. Although statistical significance is not achieved, there is a trend towards longer survival in patients whose tumors express bcl-2. This tendency is also reflected by a higher rate of complete remission after chemotherapy: 40% in patients with bcl-2+ tumors versus 27% in patients with bcl-2- tumors. In multivariate analysis, tumor stage followed by Karnofsky index were the most valuable predictors for complete remission. LDH and tumor stage were most predictive for 1-year survival. Bcl-2 expression is frequent in SCLC and may reflect a less aggressive mechanism of transformation and a higher susceptibility to cytostatic treatment.

Steroid-hormone receptors in cell lines and tumor biopsies of human lung cancer.

Female gender is a significant independent favorable prognostic factor in lung cancer. To study the possible role of sex hormones in lung cancer, the expression of sex-steroid receptors and the glucocorticoid receptor was investigated in 29 lung-cancer cell lines stemming from small-cell lung cancer (SCLC) and non-small-cell lung cancer (NSCLC) by means of immunocytochemistry, ligand-binding assays and RNA expression via polymerase chain reaction. In at least 2 methods of investigation, NSCLC cell lines showed a low expression of estrogen receptor in 6, progesterone receptor in 13 and androgen receptor in 12 out of 17 cases examined; sex-steroid-receptor expression was virtually absent in SCLC cell lines. The glucocorticoid receptor was expressed in all 29 cell lines studied. Additionally, 52 tumor samples from primary lung cancer were investigated for their receptor expression by means of immunohistochemistry. Among patients with primary lung-cancer sex-steroid-receptor expression in tumor biopsies was detected most frequently in female patients (in 69% of 16 cases, vs. 42% of 36 tumors from men) and in patients with adenocarcinoma. Further research will focus on these subgroups. Immunohistology is a feasible method of studying steroid-receptor expression in lung cancer.

Expression of vitamin D receptor in lung cancer.

The active metabolite of vitamin D 1,25-dihydroxycholecalciferol is a hormone-like agent that regulates cell differentiation and proliferation. Various vitamin D derivatives have been shown to induce differentiation in neoplastic cells. The prerequisite for any hormone action is the presence of its receptor. We studied the expression of vitamin D receptor in human lung cancer cell lines and in primary lung cancer tissue. Employing the polymerase chain reaction, 10 out of 11 cell lines stemming from small-cell lung cancer and 15 out of 15 cell lines stemming from non-small-cell lung cancer demonstrated vitamin D receptor expression. An immunohistochemical analysis, using a specific monoclonal antibody, demonstrated vitamin D receptor protein expression in 31 out of 117 (26%) primary small-cell lung cancer cases tested. Positive cells exhibited a nuclear reaction pattern. Twenty-one out of 37 primary non-small-cell lung cancer cases, particularly adenocarcinomas (9/14) and squamous-cell carcinomas (10/15), exhibited vitamin D receptor. Results indicate that a subset of lung cancer cases may be susceptible to the differentiating effects of vitamin D analogues.

Hair follicle expression of 1,25-dihydroxyvitamin D3 receptors during the murine hair cycle.

Because the hair follicle is a highly hormone-sensitive miniorgan, the role of hormones produced locally in the skin in the control of hair growth deserves systematic analysis. It has been shown previously that the potent steroid hormone 1,25-dihydroxyvitamin D3 (1,25-D3) modulates growth and differentiation of keratinocytes via binding to a high-affinity nuclear vitamin D receptor (VDR). In this study, we have examined the in situ expression of VDR during the murine hair cycle. VDR expression was detected immunohistochemically. To obtain defined stages of the murine hair cycle, hair growth was induced by depilation in C57 BL-6 mice. In addition to the recognized VDR expression of outer root sheath keratinocytes, we detected VDR immunoreactive cells in the dermal papilla, the mesenchymal key structure of the hair follicle. Furthermore, VDR immunoreactivity in the nuclei of outer root sheath keratinocytes and in dermal papilla cells was stronger during anagen IV-VI and catagen than during telogen and anagen I-III. This suggests hair cycle-associated changes in the expression of VDR, and points to a potential role for 1,25-D3 in hair follicle biology. Selected follicular cell populations may display hair cycle-dependent sensitivity to 1,25-D3 stimulation.

[Vitamin D3 and its analogs as multifunctional steroid hormones. Molecular and clinical aspects from the dermatologic viewpoint]. / Vitamin D3 und seine Analoga als multifunktionelle Steroidhormone. Molekulare und klinische Aspekte aus dermatologischer Sicht.

Since the identification of the cholesterol derivative 1,25-dihydroxy-vitamin D3 and its analogues as potent immunomodulatory, proliferation- and differentiation-regulatory molecules, the amount of data available on the effects of these agents on the skin and its appendages has grown exponentially. This review outlines recent progress in the understanding of the molecular biology and pathophysiology of vitamin D, and new strategies for the treatment of skin diseases are discussed. Focusing on psoriasis and preliminary clinical experiences, we discuss possible therapeutic targets and perspectives for these multifunctional steroid hormones in dermatology.