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Aplidine, dehydrodidemnin B, is a marine depsipeptide isolated from the Mediterranean tunicate Aplidium albicans currently in phase II clinical trial. In human Molt-4 leukaemia cells Aplidine was found to be cytotoxic at nanomolar concentrations and to induce both a G1 arrest and a G2 blockade. The drug-induced cell cycle perturbations and subsequent cell death do not appear to be related to macromolecular synthesis (protein, RNA, DNA) since the effects occur at concentrations (e.g. 10 nM) in which macromolecule synthesis was not markedly affected. Ten nM Aplidine for 1 h inhibited ornithine decarboxylase activity, with a subsequently strong decrease in putrescine levels. This finding has questionable relevance since addition of putrescine did not significantly reduce the cell cycle perturbations or the cytotoxicity of Aplidine. The cell cycle perturbations caused by Aplidine were also not due to an effect on the cyclin-dependent kinases. Although the mechanism of action of Aplidine is still unclear, the cell cycle phase perturbations and the rapid induction of apoptosis in Molt-4 cells appear to be due to a mechanism different from that of known anticancer drugs. British Journal of Cancer (2002) 86, 1510–1517. DOI: 10.1038/sj/bjc/6600265 www.bjcancer.com © 2002 Cancer Research UK

The ability of the TRAIL ligand to induce cell killing in three ovarian cancer cell lines was investigated using a glutathione-S-transferase (GST)-TRAIL fusion protein. One of the three lines was sensitive to TRAIL, which induced cell killing in a range of concentrations similar to those necessary to kill the TRAIL-sensitive leukaemic cell line Jurkat. The relative mRNA expression of the four TRAIL receptors did not explain the different sensitivities of the three ovarian cancer cell lines to TRAIL treatment. The TRAIL-sensitive IGROV-1 cell line expressed slightly lower levels of the anti-apoptotic protein FLIP than the two TRAIL-insensitive cell lines (A2780 and SKOV-3). Nevertheless, although TRAIL did not significantly reduce cell growth in the A2780 and SKOV-3 cells it did enhance the activity of paclitaxel and cisplatin (DDP), the two most widely used drugs for the treatment of ovarian cancer, increasing their ability to induce apoptosis. The use of TRAIL in combination with classical anticancer agents might thus boost the apoptotic response, improving the activity of DDP and paclitaxel in ovarian cancer.

Summary Background: The levels and activity of topoisomerase I were determined in 35 biopsies from patients with ovarian cancer. Patients and methods: The activity was denned by the ability to relax supercoiled DNA plasmid, and levels were determined by Western blotting and immunohistochemistry. Results: We detected topoisomerase I activity in all samples, although at different levels. Enzymatic activity was the same in fresh and frozen tissues. Western blotting analysis detected topoisomerase I in 29 of 35 tumor samples but in the remaining six the levels were below the detection limit. We analyzed the distribution of topoisomerase I in tumor cells and normal infiltrating cells by immunohistochemistry. The enzyme was mainly associated with tumor cells although there were four samples in which tumor cells were negative but in which normal cells, mainly lymphocytes, yielded a positive result. Conclusions: The results indicate that topoisomerase I enzymatic activity is detectable in human ovarian tumors, varying among patients. No correlations were found between the levels of the enzyme and its activity. Because of the high heterogeneity observed, enzymatic activity assays should be combined with immunohistochemical evaluation of Topo I in the tumor and normal cells present in the tissue.

The cyclin-dependent kinase inhibitor p16 gene (P16, MTS1, CDKN2) has been shown to be altered by deletion or point mutation in some human tumours and cancer cell lines, suggesting that it works as a tumour suppressor. We analysed p16 gene mutation and p16 protein expression in 42 primary ovarian carcinomas and in five human ovarian cancer cell lines. Polymerase chain reaction (PCR) amplifications of exons 1 and 2 of the gene showed no deletion or gross rearrangement in the p16 gene. The lack of deletion was further demonstrated by Southern blot analysis. Looking for point mutations, we used single-strand confirmation polymorphism (SSCP) analysis and, in half of the tumours, we sequenced both strands of exons 1 and 2. No mutations were detected. In 11 out of 42 patients (26%), however, we detected no protein expression by Western blot analysis, suggesting that decreased expression of p16 rather than deletion of the gene can occur in a significant percentage of human ovarian cancers. In the same experiment CDK4 protein was found homogeneously expressed in all the tumour specimens and in the five cell lines. The lack of expression of p16 was not due to hypermethylation of the gene assessed by digestion of genomic DNAs with a methylation sensitive enzyme, suggesting that other mechanisms, not yet identified, are involved in the decreased expression of the p16 gene in human ovarian tumours. Images Figure 1 Figure 2 Figure 3 Figure 4

The expression of different genes potentially involved in DNA repair and in cell responses to chemotherapy was evaluated in 33 previously untreated ovarian cancer patients. In biopsies of the same patients the expression of repair genes O6-methylguanine DNA methyltransferase (MGMT), 3-methyladenine DNA glycosylase (MAG), ERCC1, MDR-1, DNA topoisomerase I, DNA topoisomerase IIalpha, and glutathione S-transferase-pi (GST-pi) was assessed by Northern blot analysis. No direct statistical correlation was found between the expression of these genes and the response to chemotherapy (mainly platinum-based with or without doxorubicin and cyclophosphamide). Univariate analysis showed a weak negative correlation (P = 0.037) between the expression of ERCC1 and mortality, whereas no statistically significant correlation was found for other parameters. The MDR-1 gene encoding for the P-glycoprotein P-170 was mostly undetectable in these patients (as assessed by Northern blotting), whereas relatively high levels of MAG and MGMT were found in the majority of patients. A statistically significant correlation was found between the expression of DNA topoisomerase I and the expression of either ERCC1 (P = 0.0026) or GST-pi (P = 0.0279).

Acute noise stress decreased [3H]5-hydroxytryptamine ([3H]5-HT) uptake in synaptosomes from rat hypothalamus, hippocampus and cerebral cortex. The decrease was due to the maximum rate of [3H]5-HT uptake, which peaked 30 min after stress and partly returned to resting values within 4 h, with no changes in affinity (Km values). No changes in [3H]paroxetine binding and basal [3H]5-HT release were found in stressed rats. Tianeptine, given at the dose of 10 mg/kg 1 h before stress, counteracted the noise-induced decrease of 5-HT uptake, since it increased [3H]5-HT uptake in both resting and stressed animals, but did not prevent the rise in plasma corticosterone of stressed rats. Buspirone pretreatment had no effect on [3H]5-HT uptake in resting rats but prevented the noise-induced decrease in [3H]-HT uptake. Diazepam did not modify either the basal or the noise-induced reduction in [3H]5-HT uptake. The evidence that treatments reducing extrasynaptic 5-HT, by increasing its reuptake (tianeptine) or reducing its release (buspirone) in innervated regions are able to modify the stress-induced decrease in 5-HT uptake, further confirms the importance of serotonin in the mechanisms mediating neurochemical responses to stress.

Protoporphyrin IX (PP) and N-methylprotoporphyrin IX (N-MePP) added in vitro to liver membranes reduced dose-dependently the affinity of [3H]PK 11195 for the mitochondrial benzodiazepine receptors (MBRs), the latter being about 20 times more potent (Ki 4.5 and 0.25 microM). Preincubation of these two porphyrins with liver homogenates for 120 min at 4 degrees resulted in significant inhibition of [3H]PK 11195 binding even after repeated washings of the membranes due to the residual presence in the membranes of about 35 and 5% of PP and N-MePP, respectively. Thus, the hypothesis that an in vivo increase in the hepatic porphyrin content modifies the binding of the isoquinoline PK 11195 to the MBRs was investigated in an experimental model of protoporphyria. PP and N-MePP were allowed to accumulate in vivo through treatment with 3,5-diethoxycarbonyl-1, 4-dihydrocollidine (DDC) (100 mg/kg i.p., once), and rats were killed 5 h after treatment when hepatic porphyrin accumulation was marked (10-fold increase), PP predominating. In the liver, treatment reduced the affinity (Kd) of [3H]PK 11195 for MBRs (from 3.56 to 15.37 nM, P0.01) and the maximum number of binding sites (Bmax) (55% decrease, P0.05); the affinity (Ki) of RO 5-4864 for [3H]PK 11195 binding sites was also reduced (from 23.9 to 72.99 nM, P0.05). No significant differences were found in the brain cortex. Liver and brain diazepam binding inhibitor levels and plasma corticosterone levels were unchanged. The reduction in [3H]PK 11195 binding to MBRs in the liver of DDC-treated rats thus appears to be attributable to a specific effect of the DDC-induced formation of the two protoporphyrins; this conclusion suggests that in hepatic protoporphyria processes modulated by MBRs may be altered.

Purpose: The PRL-3 phosphatase has been found expressed at higher levels in metastasis than in primary tumors of patients with colorectal cancer. In the present study, we evaluated the expression of PRL-3 in ovarian cancer tissue and its role in ovarian cancer cell growth. Experimental Design: PRL-3 phosphatase expression was evaluated in 84 ovarian tumor samples. PRL-3 expression has been knocked down using specific small interfering RNAs to determine its role in ovarian cancer cell growth in vitro. Results: In ovarian cancers, PRL-3 expression correlates with disease progression, being higher in advanced (stage III) than in early (stage I) tumors. In situ measurements of PRL-3 expression showed that it was confined to the epithelial neoplastic cells. The molecular mechanism underlying PRL-3 overexpression in ovarian cancers is independent from amplification of the corresponding genomic locus. Ovarian cancer cells growing in culture have high levels of expression of this phosphatase. PRL-3–specific knockdown using small interfering RNA severely impaired the growth of cells without affecting the expression of the closely related homologue PRL-1. Intriguingly, the growth of human colon carcinoma cells expressing lower levels of the PRL-3 was not affected by the PRL-3 knockdown. Conclusions: Altogether, these results show that PRL-3 expression is associated with ovarian cancer progression and point to a key role for this phosphatase in the control of ovarian cancer cells growth. This strongly suggests that PRL-3 should be considered as a target for the discovery of new anticancer agents to be tested against this malignancy.

The present study aimed to establish the role of the metabolite norfluoxetine in the anorectic activity of fluoxetine, and to relate the anorectic doses (ED50) to the brain concentrations of the parent drug and its metabolite. Fluoxetine showed anorectic activity at increasing intraperitoneal doses (ED50 = 391, 34·7 and 21·7 μmol kg−1 in mouse, rat and guinea-pig, respectively) and norfluoxetine was slightly more active (24·3, 22·9 and 19·1 μmol kg−1, respectively) in all three species. In terms of maximum concentration (Cmax) and area under the curve (AUC) within the experimental period (0–90 min), brain concentrations varied widely and were poorly related to the dose; guinea-pig appeared to be much more sensitive to fluoxetine than was mouse or rat. Administered norfluoxetine was present in the brain of the three species in approximately the same order as fluoxetine, i.e. lower in guinea-pig than in mouse or rat. The Cmax and AUC of norfluoxetine after fluoxetine administration was 50–60% of the values after an equiactive dose of norfluoxetine in mouse and guinea-pig, and more than 80% in rat.

Summary Background The p73 gene is structurally related to the tumor suppressor genep53 The role of p73 in tumor development is still unclear and no data on ovarian cancer are so far available. For this reason we have analyzed, in a panel of ovarian cancers, the allelic distribution and expression of p73. Patients and methods Fifty-one samples from ovarian cancers and five human ovarian cancer cell lines growing in culture were analyzed. Allelic origin was analyzed by PCR after digestion with the restriction enzyme Sty I. Heterozygous, informative cases were selected for studies aimed at evaluating allelic expression of p73. Results We found an allelic distribution similar to that previously reported. LOH was found in two patients with ovarian cancer. In one case in which normal ovarian tissue was available biallelic expression of p73 was found. Conclusion In comparisons of ovarian cancers and borderline tumors, no differences in allelic distribution and/or expression were found, suggesting that p73 does not play an important role in the pathogenesis and development of ovarian cancer.

In a panel of 16 human ovarian tumours transplanted in nude mice, the expression of genes involved in cell cycle regulation and in response to drug treatment were characterised. In the 16 tumours analysed we could not detect overexpression of Erb-B2 oncogene while expression of MDR1 mRNA was not detected in 11/15 samples and was low in 4/15 tumours. Only three tumours had mutations in the p53 gene exons 5-8 and one of these mutations did not result in any amino acid alteration. The levels of mRNA for cyclins A, D1 and E were heterogeneous with some tumours expressing high levels and others not expressing them at all. The same was found for the cyclin dependent kinases (CDK) CDK2 and CDK4 and for CDK inhibitors p21/WAF1, p27/KIP1 and p16/CDKN2. Two genes belonging to the nucleotide excision repair, ERCC1 and ERCC3 were detectable in all the samples examined, as were the genes MGMT and MAG, also involved in DNA repair. The data indicate a heterogeneity in the expression of genes considered to be involved in the cellular responses to cytotoxic drug treatment and indicate the possibility of using these tumour models to test specifically molecules with a defined mechanism of action.

We selected two clones, isolated from the human colocarcinoma cell line LoVo, showing a sensitivity to doxorubicin similar to (LoVo clone 5) or three times lower than (LoVo clone 7) the parental cell line. Since vimentin was atypically expressed in a human breast carcinoma cell line made resistant to doxorubicin, we looked at vimentin expression in these two clones with spontaneously different sensitivity to the drug. For comparison we used the parental cell line LoVo WT and LoVo/DX made resistant pharmacologically. mRNA for vimentin was undetectable by Northern blot analysis in LoVo WT and in LoVo clone 5, while expression of this gene was high in LoVo clone 7 and in LoVo/DX. This increase in mRNA levels was not related to an amplification of DNA, as suggested by Southern blot analysis. Immunofluorescence and immunocytochemistry findings confirmed, at protein level, the mRNA data. In LoVo clones 5 and 7, there were respectively 8.6% and 71% vimentin-positive cells, although the two clones showed similar expression of multidrug resistance gene 1 (mdr-1) and accumulated intracellular doxorubicin at similar levels. Similarly, drug efflux was the same for both clones. Our results show for the first time that cells resistant to doxorubicin express vimentin independently of the mdr glycoprotein. However when cells from clone 5 were transfected with human vimentin cDNA, they did not become resistant, indicating that vimentin can be considered as a marker of resistance in these cells but does not give rise to a resistant phenotype by itself. Images Figure 1 Figure 2 Figure 3 Figure 4 Figure 5

The glutathione (GSH)-glutathione S-transferase (GST) detoxification system is an important element in cellular defence against injurious agents and anticancer drugs. GST isoenzymes may represent biochemical markers of neoplastic transformation, and, possibly, drug resistance is associated with altered GST-isoenzyme levels. The ability to measure GST-isoenzymes in cell populations would be useful for several biological and clinical applications. We have developed an immunofluorescence flow cytometric method for the simultaneous detection of different GST-isoenzymes and of DNA in fixed cells. Due to the impossibility of working under saturating conditions for the anti-GST antibody, a normalizing procedure was developed to permit quantitative analysis of single cells labelled with the anti-GST antibody at high dilution. A theoretical model and experimental data supported the use of this procedure. The method proposed is general and could be applied to other antibodies in order to obtain quantitative data outside saturating conditions. The method was challenged in different applications in order to compare it with other classical techniques. First, we characterized sublines resistant to different anticancer drugs with respect to variations of GST isotypes. In a second application, we studied the intercellular heterogeneity of GST content in mouse renal cells. In addition, GST was determined in aneuploid cells from solid tumor biopsies by separation from diploid cells on the basis of DNA content. Finally, GST distribution during cell-cycle progression was studied in two different cell lines by the biparametric analysis of GST/DNA. © 1995 Wiley-Liss, Inc.

The present study compares the anorectic activity of d-fenfluramine and its metabolite d-norfenfluramine in three animal species. d-Fenfluramine and d-norfenfluramine show anorectic activity at increasing doses (ED50) in rats, guinea pigs, and mice, d-norfenfluramine being more active than d-fenfluramine in all three species. Equiactive anorectic activities are reached with different brain levels of d-fenfluramine and d-norfenfluramine, guinea pigs being the most sensitive species, followed by rats then mice. The metabolite most probably plays a major role in the anorectic effect of d-fenfluramine in guinea pigs, contributes to the anorectic activity in rats, but adds little to the action of the parent drug in mice. The different sensitivity to d-fenfluramine and d-norfenfluramine in these three species does not appear to be explained by a number of biochemical parameters, including serotonin uptake or release, receptor subtypes, or 3H-d-fenfluramine binding and uptake.

Liquipron and Toprina, obtained by growing yeasts (Candida maltosa and Candida lipolytica) on n-hydrocarbons, were investigated to ascertain the biological significance and possible toxicological implications of their high content of uneven fatty acids (UFA). It was confirmed that the extent to which UFA accumulate in adipose tissue of rats fed the 2 products reflects only partially their UFA contents. The presence of UFA in rat tissues does not appear to alter intermediate metabolism. The capacity of liver mitochondria to oxidize palmitic acid was similar in control and in Liquipron-treated rats. Palmitic acid and heptadecanoic acid did not compete for oxidation when mixed at concentrations which reflect their presence in the tissues of animals fed high levels of Liquipron.

Adipose tissue of rats pretreated with 5-carboxy-3-methylpyrazole (5C3MP) or nicotinic acid released less glycerol and FFA (basal lipolysis) in their incubation medium. However, they were more sensitive to the lipolytic action of norepinephrine or theophylline. The maximum release of FFA and glycerol due to norepinephrine addition was increased in adipose tissue from 5C3MP pretreated rats. Moreover, the sensitivity to cyclic 3′5′ AMP (dibutyryl ester) was not significantly increased. The hypersensitivity to lipolytic agents already appeared at times when plasma FFA were lowered, but was present also during the rebound phase. Adrenalectomy did not prevent the inhibition of lipolytic activity exerted by these compounds, but almost completely inhibited the hypersensitivity of adipose tissue of treated rats to lipolytic agents. The possibility that these changes in the responsiveness of adipose tissue were correlated with the increased level of plasma corticosterone observed after the administration of 5C3MP or nicotinic acid are taken into consideration. A possible role of the hypersensitivity in the onset of rebound effect is discussed.

THE level of utilizable glucose represents a control of the release of free fatty acids (FFA) and, therefore, of the level of serum FFA1. Drugs acting on glucose utilization are therefore likely to be effective in changing the level of FFA. The observation that salicylate increases oxidative reactions2,3 led us to the finding that this drug is a powerful inhibitor of FFA release. Previous investigations carried out in man were contradictory. While Carlson4,5 found a decrease of plasma FFA in normal and diabetic subjects, Gilgore et al.6 did not confirm these results. However, it is likely that in the latter data there was an interference of salicylate in the determinations of FFA carried out according to Dole's method.

5 Carboxyl-3 methylpyrazole (5C3MP) and nicotinic acid are very effective in lowering FFA in plasma and white adipose tissue but not in brown adipose tissue under conditions of FFA mobilization obtained by fasting and cold and by the administration of norepinephrine, theophylline and ACTH. Similar results were obtained also in vitro except that in this case the effect of theophylline on brown adipose tissue was also slightly inhibited by 5C3MP. PGE1 inhibited the effect of norepinephrine and theophylline on white but not on brown adipose tissue. The results obtained indicate that in vivo the level of FFA in plasma depends more on the lipolysis occurring in the white than in the brown adipose tissue.

Summary Diazepam binding inhibitor (DBI) acts in brain by binding to GAB/benzodiazepine receptors (GBR) and to mitochondrial benzodiazepine receptors (MBR). Because DBI acting at MBR, has been shown to be an effector of ACTH-induced steroidogenesis and stress is known to change the level of GBR and MBR, the model of acute noise stress in rats was used to study modifications of DBI and GRB or the content of MBR in various areas of the brain and adrenal gland. It was found that, in the brain of stressed rats, DBI and its processing products (ODN-like immunoreactivity), increased selectively in the hippocampus. This increase in the content of DBI was preceded and followed by a net decrease of GBR and an increase of MBR. Similarly, in adrenal cortex, the content of DBI and MBR increased during the first hour, following acute stress and this increase paralleled the increase in plasma corticosterone. These data suggest that DBI, acting on MBR may regulate steroidogenic function in stress.