[Preclinical studies of oxaliplatin in combination chemotherapy]. / In process citation.

Therapeutic innovation in oncology often requires the optimization of combinations with active drugs based on in vitro and in vivo data. This is exemplified by oxaliplatin for which several preclinical studies of combinations have been realized. Oxaliplatin has been combined with 5-fluoro-uracile, gemcitabine, topoisomerase I inhibitors, taxanes demonstrating synergy or additivity. Synergistic and additive effects are often due to the optimization in the use of distinct mechanism of action or resistance and might be associated with no overlapping toxicity when combined in clinical trials.

Synergistic antitumoral activity of combined UFT, folinic acid and oxaliplatin against human colorectal HT29 cell xenografts in athymic nude mice.

This study was designed to assess the inhibition of tumor growth by oxaliplatin combined with UFT and folinic acid (FA). Growth inhibition was studied in nude mice transplanted with human colorectal HT29 tumor cell xenografts and treated for 28 days with oral UFT (20 mg/kg/day) and FA (4 mg/kg/day), i.p. oxaliplatin (10 mg/kg on day 1) or a combination of oxaliplatin, UFT and FA, or else not treated (controls). Tumor surface area and weight were recorded twice a week, and mice were sacrificed at day 28. Two separate experiments were performed for each group of 25 mice. At day 28, mean tumor weights (g) were 2.89+/-0.22 (controls), 2.03+/-0.14 (oxaliplatin), 2.02+/-0.21 (UFT/FA) and 1.23+/-0.17 (oxaliplatin+UFT/FA). For the three treatment groups, tumor weight decreases were 30.1% (p<0.05), 29.9% (p<0.05) and 57.5% (p<0.001), respectively. Combined treatment (UFT/FA+oxaliplatin) reduced tumor weight by 39% compared to oxaliplatin alone (p<0.05) or UFT/FA (p<0.05). These results demonstrate the synergistic effect of the combination of oxaliplatin, UFT and FA in this HT29 cell xenograft model, and warrant further investigations in patients with metastatic colorectal cancer.

Vegf, Vegf-B, Vegf-C and their receptors KDR, FLT-1 and FLT-4 during the neoplastic progression of human colonic mucosa.

Because the crucial role of angiogenesis has been demonstrated in tumor growth and metastasis, the present study was undertaken to characterize the relative expression of vascular endothelial growth factors VEGF (vascular endothelial growth factor), VEGF-B, VEGF-C, and their receptors KDR (kinase insert domain-containing receptor), FLT-1 (fms-like tyrosine kinase), and FLT-4 in human colonic cancers, in relation to the Astler-Coller pathological classification, and to prognosis. VEGF and VEGF-B gene expression was quantified by Northern blot in 72 tumor samples matched with control tissues. VEGF gene expression was 1.4 times higher in adenocarcinomas than in control tissues (p = 0.02), but did not increase further between Astler-Coller tumor stages A and D, and did not correlate with disease recurrence for patients at stages B2 or C. In adenomas, VEGF mRNA levels were not significantly different from those in the paired control colonic mucosa. The expression pattern of VEGF isoforms, mainly identified by RT-PCR (reverse-transcriptase-coupled polymerase chain reaction) as VEGF121 and VEGF165 and to a lesser extent VEGF189, was comparable in tumor and control tissues. VEGF-B mRNA levels were unchanged during the neoplastic progression of colonic mucosa. In contrast to KDR and FLT-4, the expression of VEGF-C and FLT-1 genes increased in some pathological tissues. These results provide evidence that the early and sustained increase in VEGF transcripts and the expression of multiple angiogenic factors and receptors contribute to the development of colon cancer, and thus constitute a putative target for anti-angiogenic drug therapy.

Devazepide (L-364718) inhibits growth and increases expression of tumor markers in HT29-S-B6 cells.

Devazepide (L-364718, a non-peptide antagonist of CCK-A receptors), inhibits the proliferation and induces morphologic changes in the mucous-secreting, autonomously proliferating human cancer colon cell line (HT29-S-B6. Addition of devazepide (10 microM) for at least 3 days in the exponential phase of growth enhanced the baseline production of gastric M1 mucins 2-3-fold and that of carcinoembryonic antigens 5-fold. Moreover, devazepide induced an increase in the amount of the MUC-5AC mRNA expressed by HT29-S-B6 cells. The increased in mucins secretion induced by devazepide was persistent after removal and independent of the presence of serum. In conclusion, devazepide-L-364718 behaves as a maturation agent in the cell clone HT29-S-B6.

Effects of ketoconazole on the proliferation and cell cycle of human cancer cell lines.

The growth-inhibitory effects of ketoconazole, an antifungal agent which inhibits arachidonic acid lipoxygenases and cytochrome P-450 enzymes, were tested in human colon and breast cancer cell lines. In the serum independent HT29-S-B6 colon cell clone, ketoconazole reduced cell proliferation and [3H]thymidine incorporation in a dose-dependent fashion, with a 50% inhibitory concentration of approximately 2.5 microM. Flow cytometry showed an accumulation of cells in the G0-G1 phase of the cell cycle and a concomitant decrease of the percentage of cells in S phase. Ketoconazole also inhibited [3H]thymidine incorporation in the hormone-independent breast cancer cells MDA-MB-231 and Evsa-T, with respective 50% inhibitory concentration of approximately 13 and 2 microM. The mechanism of action of ketoconazole is unknown. However, another lipoxygenase inhibitor, BW755C, inhibited only weakly [3H]-thymidine incorporation and accumulated the cells in S and G2. Conversely, clotrimazole and SKF525A, inhibitors of cytochrome P-450 enzymes, had effects similar to those of ketoconazole on HT29-S-B6 cells whereas metronidazole and secnidazole, other azole derivatives which do not inhibit cytochrome P-450 enzymes, had no effect. The results suggest that cytochrome P-450 enzyme(s) activity(ies) could be implicated in the antiproliferative effects of ketoconazole.

Differentiation of newborn rat preadipocytes in culture: effects of insulin and dexamethasone.

A preadipocyte cell population isolated from the inguinal tissue of 3-day-old rats converts at confluence into mature adipocytes when cultured with insulin (10(-9) M). Insulin is necessary only from Day 4 postplating. If the addition of insulin is further delayed, the proportion of cells which will undergo adipose conversion decreases. A loss of the differentiation competence is also observed when the cells are allowed to proliferate (seeding at a low density in a serum containing medium). A preexposure of the primary cells to dexamethasone during the insulin-insensitive period (Days 0-4) accelerates the subsequent "insulin-dependent" adipose conversion. In order to produce its effect, dexamethasone needs only to be present for 4 h on Day 2 postplating. The effect of dexamethasone is probably due neither to inhibition of cell proliferation nor to induction of the cell content of insulin receptors. The evolution of G3PDH enzyme activity as well as of G3PDH protein and mRNA was used as an indicator of the differentiation process. The enzyme accumulates to a low extent during culture in the absence of insulin. When insulin is present, the enzyme level is dramatically increased (maximum on Day 11). Dexamethasone pretreatment (Days 0-4, or 4 h on Day 2) accelerated the G3PDH enzyme activity increase as well as protein and mRNA accumulation. This was also true in cells maintained in insulin-free medium; however, in this case, the increase in the enzyme activity was limited to the first 8 days of culture and full differentiation did not take place. We conclude that: (1) the rat preadipocytes are committed to differentiate, requiring insulin as a sufficient physiological stimulus; (2) the differentiation program is progressively lost after greater than 4 days of culture without insulin and more rapidly if the cells are allowed to undergo divisions; and (3) dexamethasone accelerates the insulin-dependent adipose conversion but alone does not ensure the complete differentiation process.

Proliferation of the human colon carcinoma cell line HT29: autocrine growth and deregulated expression of the c-myc oncogene.

Human colon adenocarcinoma cell (line HT29) are able to proliferate in a defined (serum-free) medium containing no added growth factors; in such conditions, their doubling time is 3 to 4 days (on serum-coated dishes) or 2 to 3 days (on an autologous extracellular matrix) compared with 1 day in the presence of fetal calf serum. In the presence of suramin, a polyanion disrupting the binding of growth factors to their receptors, the incorporation of [3H]thymidine in serum-free cultures is reduced (27.0 +/- 2.9% of control after 3 days of culture), suggesting involvement of autocrine growth factors in the autonomous proliferation of the cells. The expression of the proliferation-related oncogene c-myc was examined during various stages of growth and differentiation of the HT29 cells. The cellular contents of c-myc mRNA were similar in all experimental conditions studied: exponential phase; stationary phase; nondifferentiated as well as differentiated cells (by glucose deprivation); and also in serum-free medium containing or not suramin. An approximately 2-fold increase in the level of c-myc mRNA was observed in cells cultured for 3 days in suramin-containing medium and then incubated during 3 h in the absence of suramin (with or without 10% fetal calf serum). Southern blot analysis of the genomic DNA of HT29 cells did not reveal any rearrangement within the region containing the c-myc gene and the flanking sequences (approximately five kilobases upstream and approximately three kilobases downstream). The c-myc locus was weakly amplified (four to six copies per cell). These results indicate that the c-myc gene expression in HT29 cells is deregulated and does not require growth factor stimulation. The deregulation of the c-myc gene may be related to the reduced growth factor requirement of the HT29 cell line.

Isolation of glucagon-37 (bioactive enteroglucagon/oxyntomodulin) from porcine jejuno-ileum. Isolation of the peptide.

A 37 amino acid-peptide has been isolated from porcine jejuno-ileum on the basis of its glucagon-like activity in liver (interaction with glucagon-binding sites and activation of adenylate cyclase) using gel filtration, ion-exchange and high-performance liquid chromatography. Depending on the criteria chosen, this peptide is referred to as either 'bioactive enteroglucagon' (activity in liver), 'oxyntomodulin' (specific action in gastric oxyntic glands) or 'glucagon-37' (chemical structure).

[Bioactive "enteroglucagon" (oxyntomodulin): evidence for a C-terminal extension of the glucagon molecule]. / "Entéroglucagon" bioactif (oxyntomoduline) : évidence pour une extension C-terminale de la molécule de glucagon.

The bioactive fraction of "enteroglucagon" has been isolated from the porcine jejuno-ileum on the basis of its glucagon-like action in liver. High performance liquid chromatography analysis followed by Dansylation of peptide fragments obtained after enzymatic digestion of both this peptide and glucagon suggests that the bioactive "enteroglucagon" contains the glucagon molecule plus a C-terminal extension under the from of the octapeptide. Lys-Arg-Asn-Lys-Asn-Asn-Ile-Ala-COOH. The presence in the native molecular of a structural difference of modification as compared to glucagon in the 1-11 fragment is possible.

"Enterolglucagon': a specific effect on gastric glands isolated from the rat fundus. Evidence for an "oxyntomodulin' action.

A newly isolated porcine glucagon-like biologically active intestinal peptide ("enteroglucagon') has been tested for its ability to stimulate the cyclic AMP system present in different tissue preparations from the rat: membranes prepared from liver or from isolated fundic glands as well as intact glands isolated from either the fundus or the antrum. Enteroglucagon (EG) was about 10 times less potent than pancreatic glucagon (G) in liver membranes, whereas it was about 20 times more potent than G in fundic membranes as well as in fundic glands, where it acts at doses as low as 3 X 10(-10) M. EG and G were practically ineffective in antral glands. It is concluded that EG is an "under-glucagon' in rat liver, whereas it is a "super-glucagon' in rat fundic glands. Accordingly, we propose to call this peptide "oxyntomodulin'.

Bioactive enteroglucagon (oxyntomodulin): present knowledge on its chemical structure and its biological activities.

A bioactive form of enteroglucagon has been isolated from porcine jejuno-ileum according to its glucagon-like effect in liver. Enzymatic digestion followed by HPLC, dansylation and partial sequence analysis strongly suggests that this peptide contains the glucagon molecule (1--29) elongated at the C-terminal end by the octapeptide Lys-Arg-Asn-Lys-Asn-Ile-Ala-COOH and possibly modified in the N-terminal region. A specific action of bioactive enteroglucagon, increase in cAMP, has been found in the acid-secreting fundic part of the rat stomach. The term "oxyntomodulin" is therefore proposed to describe this peptide.