Effects of alvocidib and carboplatin on ovarian cancer cells in vitro.

AIM: Failure of platinum chemotherapy is an unresolved issue in ovarian cancer. Targeted therapy has been added to the treatment options in solid cancers. Alvocidib is a cyclin dependent kinase inhibitor. MATERIALS AND METHODS: This study evaluated the effects of alvocidib together with carboplatin on ovarian cancer cells (BG-1 and Skov-3) in vitro applying proliferation assays, cell cycle distribution analyses, apoptosis induction assays, and drug accumulation assay. RESULTS: Proliferation of both cell lines was inhibited by carboplatin and alvocidib. The interaction index revealed drug synergism at distinct drug concentrations. Cell cycle distribution was altered. Alvocidib induced apoptosis in Skov-3 cells, and necrosis in BG-1 cells. Rhodamine accumulation was increased by alvocidib or both compounds together. CONCLUSION: These data provide evidence for antiproliferative effects of alvocidib on human ovarian cancer cells in vitro associated with changes in cell cycle distribution, the induction of apoptosis, and modulation of intracellular drug accumulation. Alvocidib and carboplatin showed some cooperative activity.

A phase II trial (AGO 2.11) in platinum-resistant ovarian cancer: a randomized multicenter trial with sunitinib (SU11248) to evaluate dosage, schedule, tolerability, toxicity and effectiveness of a multitargeted receptor tyrosine kinase inhibitor monotherapy.

BACKGROUND: Recurrent platinum-resistant ovarian cancer usually has a poor outcome with conventional chemotherapeutic therapy and new treatment modalities are warranted. This phase II study was conducted to evaluate sunitinib, an oral antiangiogenic multitargeted tyrosin kinase inhibitor, in this setting. MATERIAL AND METHODS: The primary end point of this randomized phase II trial was the objective response rate according to RECIST criteria and/or Gynecologic Cancer InterGroup CA125 response criteria to sunitinib in patients with recurrent platinum-resistant ovarian cancer who were pretreated with up to three chemotherapies. A selection design was employed to compare two schedules of sunitinib (arm 1: 50 mg sunitinib daily orally for 28 days followed by 14 days off drug; and arm 2: 37.5 mg sunitinib administered daily continuously). RESULTS: Of 73 patients enrolled, 36 patients were randomly allocated to the noncontinuous treatment arm (arm 1) and 37 patients were randomly allocated to the continuous treatment arm (arm 2). The mean age was 58.8 and 58.5 years, respectively. We observed six responders (complete response + partial response) in arm 1 (16.7%) and 2 responders in arm 2 (5.4%). The median progression-free survival (arm 1: 4.8 [2.9-8.1] months; arm 2: 2.9 [2.9-5.1] months) and the median overall survival (arm 1: 13.6 [7.0-23.2] months; arm 2: 13.7 [8.4-25.6] months) revealed no significant difference. Adverse events included fatigue as well as cardiovascular, gastrointestinal and abdominal symptoms, hematologic and hepatic laboratory abnormalities. Pattern and frequency of adverse events revealed no substantial differences between both treatment groups. CONCLUSIONS: Sunitinib treatment is feasible and moderately active in relapsed platinum-resistant ovarian cancer. The noncontinuous treatment schedule should be chosen for further studies in ovarian cancer.

Dietary omega-3, omega-6, and omega-9 unsaturated fatty acids and growth factor and cytokine gene expression in unstimulated and stimulated monocytes. A randomized volunteer study.

Dietary omega-3 fatty acids retard coronary atherosclerosis. Previously, we demonstrated that dietary omega-3 fatty acids reduce platelet-derived growth factor (PDGF)-A and PDGF-B mRNA levels in unstimulated, human mononuclear cells (MNCs). In a randomized, investigator-blinded intervention trial, we have now compared the effect of ingestion of 7 g/d omega-3, omega-6, or omega-9 fatty acids for 4 weeks versus no dietary intervention on PDGF-A, PDGF-B, heparin-bound epidermal growth factor (HB-EGF), monocyte chemoattractant protein-1 (MCP-1), and interleukin-10 gene expression in unstimulated MNCs and in monocytes that were adherence-activated ex vivo in a total of 28 volunteers. In unstimulated MNCs, mRNA steady-state levels of PDGF-A, PDGF-B, and MCP-1 were reduced by 25+/-10%, 31+/-13%, and 40+/-14%, respectively, after omega-3 fatty acid ingestion, as assessed by quantitative polymerase chain reaction (all P<0.05). In monocytes that were adherence-activated ex vivo for 4 and 20 hours, mRNA steady-state levels of PDGF-A, PDGF-B, and MCP-1 were reduced by 25+/-13%, 20+/-15%, and 30+/-8%, respectively (all P<0.05). Interleukin-10 and HB-EGF mRNA steady-state levels were not influenced by omega-3 fatty acid ingestion. Expression of all respective mRNAs in control volunteers or in those ingesting omega-6 or omega-9 fatty acids were not altered. We conclude that human gene expression for PDGF-A, PDGF-B, and MCP-1, factors thought relevant to atherosclerosis, is constitutive, is constant, and can be reduced only by dietary omega-3 fatty acids in unstimulated and adherence-activated monocytes.

Nitric oxide downregulates tumour necrosis factor in mRNA in RAW 264.7 cells.

Nitric oxide (NO) and tumour necrosis factor (TNF) are essential mediators in a number of biological processes, including the immune response. TNF stimulates NO production via expression of inducible NO synthase (iNOS), with L-arginine being the only substrate. Previously, we demonstrated that, inversely, NO inhibits lipopolysaccharide (LPS)-induced TNF synthesis. We have now investigated whether this reduction of TNF bioactivity is also reflected at the level of TNF mRNA in the murine macrophage cell line RAW 264.7. TNF mRNA was quantified by Northern analysis using an alpha[33P]dCTP-labelled probe. Cells stimulated with 10 microg/ml LPS in the absence of L-arginine, in order to prevent endogenous NO formation, contained more TNF mRNA than cells supplied with 1 mM L-arginine at 14 h and 20 h after stimulation. By contrast, no difference was observed at 4 h. This time course is compatible with the involvement of iNOS. The half-life of TNF mRNA in the presence of NO was roughly half that observed under L-arginine-free conditions (41 min versus 77 min, respectively). L-citrulline (1 mM), which has been shown to be recycled in RAW 264.7 cells to L-arginine, completely restored attenuation of TNF bioactivity and TNF message to control levels obtained with 1 mM L-arginine. Together, these findings suggest that endogenous NO regulates TNF mRNA, mainly by reducing its half-life. In addition, a distinct additional band (approximately 1.4 kb) hybridizing with the TNF probe was consistently observed in non-stimulated cells. This may correspond to TNF mRNA specifically hydrolysed at the AU-rich region, possibly reflecting another control point for TNF expression.

Anti-inflammatory activities of cAMP-elevating agents: enhancement of IL-10 synthesis and concurrent suppression of TNF production.

Interleukin-10 (IL-10) and tumor necrosis factor (TNF) exert key roles in some acute and chronic inflammatory diseases. In this study we investigated (1) the potency of different cAMP-elevating agents in enhancing IL-10 synthesis, (2) the involvement of protein kinase A in this enhancement, and (3) the mutual dependence of cAMP-enhanced IL-10 formation and cAMP-suppressed TNF synthesis. Rolipram, a specific phosphodiesterase inhibitor and cicaprost, a prostacyclin analogue, were applied as cAMP-elevating agents. The stable cAMP antagonist (Rp)-cAMPS was used to abrogate activation of protein kinase A. Human peripheral blood mononuclear cells were stimulated with lipopolysaccharide (LPS). TNF was quantified by radioimmunoassay, IL-10 by enzyme-linked immunosorbent assay, and mRNA by reverse transcriptase-polymerase chain reaction. After LPS stimulation alone 253+/-45 pg/mL IL-10 was synthesized, which increased to 644+/-117 pg/mL in the presence of 1 microM rolipram. (Rp)-cAMPS reversed this increase of IL-10 formation. In the same samples, the LPS-stimulated production of TNF was markedly attenuated by rolipram or cicaprost. A kinetic analysis revealed a significant increase in TNF production before IL-10 formation was detectable. These results demonstrate that (1) cAMP-elevating agents enhance IL-10 synthesis and suppress TNF production; (2) these regulative functions of cAMP-elevating agents are mediated by activation of protein kinases A; (3) suppression of TNF synthesis by cAMP in the early phase is not mediated by endogenous IL-10. Taken together, rolipram and cicaprost exert a dual regulatory function by enhancing IL-10 formation and attenuating TNF synthesis.

Developmental regulation of presenilin-1 processing in the brain suggests a role in neuronal differentiation.

Most cases of early-onset familial Alzheimer's disease are caused by mutations in the presenilin genes. Presenilin-1 (PS1) is subject to proteolytic cleavage resulting in the accumulation of N- and C-terminal fragments. In this report, we show that the proteolytic cleavage of PS1 is developmentally regulated in the brain. Low levels of full-length PS1 and higher levels of 30-kDa N-terminal and 20-kDa C-terminal fragments are identified at all developmental stages in the rat brain. However, in the adult brain, additional 36-kDa N-terminal and 14-kDa C-terminal fragments appear and become major PS1 species. Alternative N-terminal PS1 fragments also appear in the adult human brain, but are more heterogenous than in the rat brain. The alternative PS1 fragments are not detected at significant levels in rat or human peripheral tissues that express PS1. The alternative cleavage of PS1 is also detected in primary cultures of rat hippocampal neurons, but not in astrocytes, and is induced by neuronal differentiation. Furthermore, alternative PS1 cleavage is detected in rat PC12 cells and human neuroblastoma SH-SY5Y cells following induction of neuronal differentiation. These results suggest that an alternative pathway of PS1 proteolytic processing is induced in the brain by neuronal differentiation. PS1 may therefore play an important role in brain development and neuronal function, which may relate to the brain-specific pathological effects of PS1 mutations.

Characterization of binding sites for a GnRH-agonist (buserelin) in human breast cancer biopsies and their distribution in relation to tumor parameters.

Gonadotropin-releasing hormone analogs (GnRH-A) have been added to the armentarium in the therapy of hormone-dependent breast cancer in premenopausal women. The effect of chronic GnRH-A-treatment in premenopausal women is based on the suppression of the hypothalamus-pituitary-ovarian axis and the reduction of sex-steroid serum levels. In addition, a number of experimental and clinical data have been accumulated indicating a direct action of GnRH-A on breast cancer cells and tissue. In this study we analyzed 235 human breast cancer biopsies for specific GnRH-A-binding. We demonstrate high affinity GnRH-A binding sites in human breast cancer tissues. The evaluation of clinical data showed no correlation of the level of GnRH-A-binding with classical tumor parameters.