Expression of cyclooxygenase 2 in human malignant melanoma.

Cyclooxygenase (COX)-2 is an inducible enzyme involved in production of prostaglandins in inflammatory processes. There is now increasing evidence that a constitutive expression of COX-2 plays a role in development and progression of malignant epithelial tumors. In the present study we investigated expression and function of COX-2 in malignant melanoma. Expression of COX-2 was determined by immunohistochemistry in 28 cases of primary skin melanoma and 4 benign nevi. We show that COX-2 was expressed in 26 cases (93%) of melanomas, with a moderate to strong expression in 19 cases (68%). Benign nevi as well as normal epithelium were negative in all cases. A constitutive expression of COX-2 mRNA and protein was found in five melanoma cell lines (A375, MeWo, SK-Mel-13, SK-Mel-28, and IGR-37) by using Northern blot as well as immunoblotting. All melanoma cell lines produced prostaglandin (PG) E2 between 468 and 3500 pg/ml as determined by ELISA. Treatment with NS-398 (50 microM), a specific inhibitor of COX-2, suppressed PGE2 production of all melanoma cell lines by 50-96%. The IC50 for inhibition of PGE2 production by NS-398 was determined as 4 microM, indicating that NS-398 acts via inhibition of the COX-2 isoenzyme. We could show that proliferation of melanoma cell lines was not influenced by treatment with NS-398 in concentrations up to 100 microM. However, NS-398 reduced Matrigel invasion of all five malignant melanoma cell lines by 50-68%. Our results indicate that COX-2 is expressed in malignant melanomas and may be involved in regulation of melanoma invasion. It remains to be investigated whether selective inhibitors of COX-2 might be useful for prevention or treatment of malignant melanoma.

Proteasome inhibitors induced caspase-dependent apoptosis and accumulation of p21WAF1/Cip1 in human immature leukemic cells.

The 26S proteasome is a non-lysosomal multicatalytic protease complex for degrading intracellular proteins by ATP/ubiquitin-dependent proteolysis. Tightly ordered proteasomal degradation of proteins critical for cell cycle control implies a role of the proteasome in maintaining cell proliferation and cell survival. In this study, we demonstrate that cell-permeable proteasome inhibitors, lactacystin, benzyloxycarbonyl(Z)-leucyl-leucyl-leucinal (ZLLLal; MG-132) and 4-hydroxy-5-iodo-3-nitrophenylacetyl-leucyl-leucyl-leucine vinyl sulfone (NLVS), induce apoptosis abundantly in p53-defective leukemic cell lines CCRF-CEM, U937 and K562 as well as in myelogenic and lymphatic leukemic cells obtained from adult individuals with relapsed acute leukemias. Leukemic cell apoptosis induced by the proteasome inhibitors was dependent on activation of caspase-3 and related caspase family proteases, because caspase-3 inhibitor N-acetyl-L-aspartyl-L-glutamyl-L-valyl-L-aspartal (Ac-DEVD-cho) and, more effectively, the general caspase-inhibitor N-benzyloxycarbonyl-L-valyl-L-alanyl-L-aspartate fluoromethylketone (Z-VAD-fmk) were capable of blocking apoptosis induced by lactacystin, ZLLLal or NLVS. Induction of apoptosis by lactacystin or ZLLLal was accompanied by cell cycle arrest at G2/M phase and by accumulation and stabilization of cyclin-dependent kinase inhibitor p21WAF1/Cip and tumor suppressor protein p53. A role of p53 in mediating apoptosis or induction of p21WAF1/Cip1 was ruled out since CCRF-CEM and U937 cells express non-functional mutant p53, and K562 cells lack expression of p53. Viability and hematopoietic outgrowth of human CD34+ progenitor cells treated with lactacystin were slightly reduced, whereas treatment of CD34 + cells with ZLLLal or the cytostatic drugs doxorubicin and gemcitabine resulted in markedly reduced viability and hematopoietic outgrowth. These results demonstrate a basic role of the proteasome in maintaining survival of human leukemic cells, and may define cell-permeable proteasome inhibitors as potently anti-leukemic agents which exhibit a moderate hematopoietic toxicity in vitro.

Suppression of the reactive oxygen intermediates production of human macrophages by colorectal adenocarcinoma cell lines.

Although some in vitro studies indicate that macrophages exert cytotoxic responses against tumour cells by production of reactive oxygen intermediates (ROI), no obvious impairment of tumour cell growth is visible in various human malignant tumours, which contain a large number of tumour-associated macrophages (TAM). We made use of an in vivo-like co-culture model of multicellular tumour spheroids of three colon carcinoma cell lines (HRT-18, HT-29, CX-2) and three functionally different phenotypes of human macrophages (27E10, RM3/1, 25F9) to investigate if tumour cells deactivate macrophage cytotoxicity. The production of ROI was measured by a lucigenin-amplified chemiluminescence assay in a 96-well-microplate luminometer. Different capabilities to produce ROI by different macrophage phenotypes were observed. However, independent of the macrophage phenotype and the tumour cell type a significant inhibition of ROI formation was found in co-cultures after 1 hr, 1 and 2 days. Macrophages were also suppressed by tumour cell supernatants, which contained anti-inflammatory cytokines transforming growth factor-beta1 (TGF-beta1) and negligible levels of interleukin-4 (IL-4) and IL-10 as shown by enzyme-linked immunosorbent assay (ELISA). Although recombinant human cytokines TGF-beta1, IL-10 and IL-4 inhibited the production of ROI in freshly isolated monocytes, these cytokines had no effect on differentiated macrophage phenotypes, indicating that these cytokines are not involved in mediating tumour-induced suppression of ROI production by human macrophages.

Induction of apoptosis in the centre of multicellular tumour spheroids of colorectal adenocarcinomas--involvement of CD95 pathway and differentiation.

Multicellular tumour spheroids (MCTS) are three-dimensional cell culture systems which are widely used in cancer research. They are characterized by an outer zone of proliferating cells, an inner region of differentiating quiescent cells and an area of so-called necrotic cell death in their centre. The exact cause of this cell death, a controversy for many years, was the aim of the present study. Our data show that cell death in the centre of MCTS of three colorectal adenocarcinoma cell lines (HRT-18, HT-29 and CX-2) was induced by apoptosis. Apoptotic cells were initially distributed at random but accumulated very quickly in the quiescent and central area at day 4-5, suggesting a time- rather than size-dependent synchronization of apoptosis parallel to the formation of the proliferation gradient in MCTS. To study mechanisms inducing apoptosis, the Fas-pathway was investigated. A cell-cell contact-dependent expression of CD95 was found in all MCTS. FasL was not detected in monolayer cultures, but was expressed in spheroids of HRT-18 and CX-2. We found that TNF alpha and TGF beta 1 activated the CD95 pathway in all three cell lines. Since both TNF-alpha and TGF-beta are known to be inducible by hypoxia in a variety of cell types, we suggest that these hypoxia-induced factors sensitize the CD95 pathway in the quiescent area of MCTS. Furthermore, a loss of the heat shock proteins 27, 32, 60, 73 and 90 was observed in the quiescent area of spheroids. This suggests that tumour cell differentiation in the inner region of MCTS may be an additional factor inducing apoptosis.

[Use of sultopride in a case of neuroleptic-induced late dyskinesia]. / Utilisation du sultopride dans un cas de dyskinésie tardive des neuroleptiques.

The observation we describe exemplifies the effectiveness of sultopride in a case of neuroleptic-induced late dyskinesia. The occurrence of dyskinesia in the course of bipolar manic-depressive psychosis parallels the administration and withdrawal of sultopride. This finding led us to extend the use of sultopride to cases of late dyskinesia due to neuroleptics.