Foxo1 is a downstream effector of Isl1 in direct pathway striatal projection neuron development within the embryonic mouse telencephalon.

Recent studies have shown that the LIM-homeodomain transcription factor Isl1 is required for the survival and differentiation of direct pathway striatonigral neurons during embryonic development. The downstream effectors of Isl1 in these processes are presently unknown. We show here that Foxo1, a transcription factor that has been implicated in cell survival, is expressed in striatal projection neurons (SPNs) that derive from the Isl1 lineage (i.e. direct pathway SPNs). Moreover, Isl1 conditional knockouts (cKOs) show a severe loss of Foxo1 expression at E15.5 with a modest recovery by E18.5. Although Foxo1 is enriched in the direct pathway SPNs at embryonic stages, it is expressed in both direct and indirect pathway SPNs at postnatal time points as evidenced by co-localization with EGFP in both Drd1-EGFP and Drd2-EGFP BAC transgenic mice. Foxo1 was not detected in striatal interneurons as marked by the transcription factor Nkx2.1. Conditional knockout of Foxo1 using Dlx5/6-CIE mice results in reduced expression of the SPN marker Darpp-32, as well as in the direct pathway SPN markers Ebf1 and Zfp521 within the embryonic striatum at E15.5. However, this phenotype improves in the conditional mutants by E18.5. Interestingly, the Foxo family members, Foxo3 and Foxo6, remain expressed at late embryonic stages in the Foxo1 cKOs unlike the Isl1 cKOs where Foxo1/3/6 as well as the Foxo1/3 target Bach2 are all reduced. Taken together, these findings suggest that Foxo-regulated pathways are downstream of Isl1 in the survival and/or differentiation of direct pathway SPNs.

Manumycin inhibits ras signal transduction pathway and induces apoptosis in COLO320-DM human colon tumour cells.

The aim of the present study was to assess the cytotoxicity of manumycin, a specific inhibitor of farnesyl:protein transferase, as well as its effects on protein isoprenylation and kinase-dependent signal transduction in COLO320-DM human colon adenocarcinoma which harbours a wild-type K-ras gene. Immunoblot analysis of isolated cell membranes and total cellular lysates of COLO320-DM cells demonstrated that manumycin dose-dependently reduced p21 ras farnesylation with a 50% inhibitory concentration (IC50) of 2.51 +/- 0.11 microM and 2.68 +/- 0.20 microM, respectively, while the geranylgeranylation of p21 rhoA and p21rap1 was not affected. Manumycin dose-dependently inhibited (IC50 = 2.40 +/- 0.67 microM) the phosphorylation of the mitogen-activated protein kinase/extracellular-regulated kinase 2 (p42MAPK/ERK2), the main cytoplasmic effector of p21ras, as well as COLO320-DM cell growth (IC50 = 3.58 +/- 0.27 microM) without affecting the biosynthesis of cholesterol. Mevalonic acid (MVA, 100 microM), a substrate of the isoprenoid synthesis, was unable to protect COLO320-DM cells from manumycin cytotoxicity. Finally, manumycin 1-25 microM for 24-72 h induced oligonucleosomal fragmentation in a dose- and time-dependent manner and MVA did not protect COLO320-DM cells from undergoing DNA cleavage. The present findings indicate that the inhibition of p21ras processing and signal transduction by manumycin is associated with marked inhibition of cell proliferation and apoptosis in colon cancer cells and the effect on cell growth does not require the presence of a mutated ras gene for maximal expression of chemotherapeutic activity.

Ultrastructural and biochemical evidence of apoptosis induced by a novel inhibitor of protein geranylgeranylation in human MIA PaCa-2 pancreatic cancer cells.

Analogs of geranylgeranyl diphosphate (GGdP) have been demonstrated to inhibit the geranylgeranylation of proteins, producing cytotoxic activity in human prostate cancer cells. A detailed study is reported on the programmed cell death in vitro of human exocrine pancreas cancer cells (MIA PaCa-2) induced by the most active compound of this series of geranylgeranylation inhibitors, the dipotassium salt of (E,E,E)[2-oxo-2-[[(3,7,11,15-tetramethyl-2, 6,10,14-hexadecatetraenyl)-oxy]amino]ethyl] phosphonic acid (BAL 9504), using transmission and scanning electron microscopy (SEM). The results show that, after 72 h of treatment with BAL 9504, 25 microM, most MIA PaCa-2 cells display the typical morphological features of apoptosis, including condensation of nuclear chromatin, dilation of endoplasmic reticulum, and fragmentation of both nucleus and cytoplasm, giving rise to small membrane-bound vesicles (apoptotic bodies); surface protrusions and blebs are well demonstrated by SEM. The electrophoresis showed the presence of various bands corresponding to fragmented DNA of 180 base pairs, or multiples of this length, thus indicating that BAL 9504 effectively induces apoptosis. The present study provides the first evidence that inhibition of protein geranylgeranylation produces apoptosis in human MIA PaCa-2 exocrine pancreas cancer cells.

Inhibition of experimental angiogenesis by the somatostatin analogue octreotide acetate (SMS 201-995).

The present study investigates the effect of the somatostatin analogue octreotide acetate (SMS 201-995) on experimental angiogenesis in vitro and in vivo. Octreotide reduced the proliferation of human HUV-EC-C endothelial cells (mean, -45.8% versus controls at 10(-9) M; P < 0.05) as well as the density of the vascular network of the chick chorioallantoic membrane (mean, -35.7% versus controls at 50 microgram; P < 0.05). Furthermore, octreotide significantly inhibited chick chorioallantoic membrane neovascularization by the human MCF-10Aint-2 mammary cells secreting the angiogenic protein FGF-3. The proliferation of endothelial and smooth muscle cells from rat aorta explants on fibronectin was reduced by octreotide 10(-8) M (mean, -32.6% versus controls; P < 0.05), and a similar effect was produced on cells sprouting from explants cultured in fibrin (mean, -52.9% versus controls; P < 0.05). Topical administration of octreotide 10 microgram/day for 6 days inhibited rat cornea neovascularization induced by AgNO3/KNO3 (mean, -50.6% versus controls; P < 0.05). Octreotide 40 microgram/day i.p was tested on angiogenesis in rat mesentery obtained by i.p. injections of compound 48/80, a mast cell degranulating agent, or conditioned medium from MCF-10Aint-2 cells and was able to reduce the extent of neovascularization (mean, -45.6 and -64.1%, respectively, versus controls; P < 0.05). These data provide evidence that octreotide is an inhibitor of experimental angiogenesis in vitro and in vivo.

Phenylacetate inhibits protein isoprenylation and growth of the androgen-independent LNCaP prostate cancer cells transfected with the T24 Ha-ras oncogene.

The refractoriness of prostate cancer to androgen suppression is the landmark of clinically aggressive disease. In this study, the androgen-dependent LNCaP prostate cancer cells were transfected with the mutated c-Ha-ras gene from the T24 human bladder cancer. The derivative clone overexpressing T24-ras (LNCaP(T24-ras)) proliferated in androgen-depleted medium and showed increased growth. Protein isoprenylation and p21ras farnesylation in LNCaP(T24-ras) cells were tested in the presence of phenylacetate to document a possible relationship with the drug-induced inhibition of cell proliferation. Phenylacetate is a differentiation inducer that down-regulates in vitro the expression of the myc oncogene and activates the human peroxisome proliferator-activated nuclear receptor involved in cell growth regulation. The drug inhibited protein isoprenylation and p21ras farnesylation in LNCaP(T24-ras) cells; IC50 values were 3.1 and 3.3 mM, respectively, compared with controls. The drug reduced the cellular levels of endogenous farnesyl-PP (mean IC50 = 3.5 mM) and inhibited activation of the p21ras downstream target, p42(MAPK)/ERK2. LNCaP(T24-ras) was more sensitive than the parental line to both growth inhibition (mean IC50 = 3.01 and 7.1 mM, respectively) and apoptosis by phenylacetate. Exogenous farnesyl- and geranylgeranyl-PP indeed reduced the effects of the drug on proliferation and apoptosis in LNCaP(T24-ras) cells. In conclusion, the inhibition of protein isoprenylation and p21ras farnesylation by phenylacetate resulted in increased chemosensitivity of the androgen-independent LNCaP(T24-ras) cells compared with LNCaP, and this effect might contribute to the pharmacological activity of the drug.

Clinical and experimental pharmacokinetic interaction between 6-mercaptopurine and methotrexate.

Clinical and experimental pharmacokinetic interaction between 6-mercaptopurine (6-MP) and methotrexate (MTX) was investigated in patients as well as in rats and in HL-60 human leukemic cells. Ten children affected by acute lymphoblastic leukemia (ALL) in remission received daily doses of 6-MP given at 25 mg/m2 and i.v. infusion of high-dose MTX at 2 or 5 g/m2 once every other week. When 6-MP was given alone, the mean peak plasma concentration (Cmax) and area under the curve (AUC) of 6-MP were 72.5 ng/ml and 225.3 h ng ml(-1). Concurrent treatment with MTX at 2 or 5 g/m2 resulted in a mean increase of 108% and 121% in the Cmax and of 69% and 93% in the AUC, respectively. In rats treated with an oral dose of 6-MP at 75 mg/m2, MTX given i.p. at 5 g/m2 produced mean increases of 110% and 230% in the Cmax and AUC of 6-MP, respectively. In HL-60 human leukemic cells incubated with 6-MP at 250 ng/ml, the cumulative intracellular concentration of 6-thioguanine and 6-MP nucleotides was not significantly modified by treatment with 20 micrograms/ml of MTX. The present findings indicate that high-dose MTX enhances the bioavailability of 6-MP as evidenced by the observed increases in the plasma Cmax and AUC of 6-MP in humans and animals.

Inhibitory effect of the somatostatin analogue SMS 201-995 and cytokines on the proliferation of human colon adenocarcinoma cell lines.

The activity of the synthetic somatostatin analogue SMS 201-995 was investigated in vitro on the growth of SW480 and SW620 human colon adenocarcinoma cell lines. The inhibition of cell proliferation was significant in SW480 cells (-19.6 +/- 1.4% at SMS 201-995 10-9 M, P < 0.05), but not in SW620 cells (-5.5 +/- 0.8% at SMS 201-995 10-8 M) as compared to untreated cultures. Moreover, SMS 201-995 10-8 M decreased the mitogenic effect of epidermal growth factor (EGF) on the SW480 cell line (-26.6 +/- 3.4% vs. cells exposed to EGF 10 ng ml-1 alone, P < 0.05). The effect of combining SMS 201-995 plus the cytokines interleukin-2 (IL-2) or gamma-interferon (gamma-IFN) on SW480 and SW620 cancer cell growth was also evaluated. The treatment produced a synergistic antiproliferative effect against SW620 cells as compared to untreated cultures, with growth inhibition being -20.2 +/- 1.2 and -19.3 +/- 1.3%, at SMS 201-995 10-8 M plus IL-2 or gamma-IFN 100 IU ml-1, respectively, but did not increase the activity of SMS 201-995 against the SW480 cells. In conclusion, the effect of SMS 201-995 on colon cancer cell growth can be enhanced by its combination with cytokines in SW620 but not in SW480 colon adenocarcinoma cells.

Plasma and tissue disposition of paclitaxel (taxol) after intraperitoneal administration in mice.

The pharmacokinetics of single intraperitoneal doses of paclitaxel (18 and 36 mg/kg) in mice were investigated in the present study. The analysis of drug concentrations by HPLC indicated that the plasma Cmax (13.0 +/- 3.1 and 25.7 +/- 2.8 micrograms/ml, respectively) were reached at the 2nd hr. The values of CL were low (0.06 and 0.1 ml/min, respectively), and t1/2 beta values of 3.0 and 3.7 hr were found, after 18 and 36 mg/kg, respectively. The highest tissue concentrations were observed in the liver (50.2 +/- 3.1 and 92.0 +/- 9.5 micrograms/g respectively), followed by the pancreas (39.3 +/- 9.9 micrograms/g) and the ovary (53.4 +/- 5.6 micrograms/g) after 18 and 36 mg/kg, respectively. In the case of the colic tissue, paclitaxel Cmax were 14.4 +/- 0.8 and 32.8 +/- 3.5 micrograms/g at the 3rd hr, respectively, with sustained drug levels still detectable 24 hr after treatment. Paclitaxel Cmax values of 12.7 +/- 3.0 and 53.4 +/- 5.6 micrograms/g were detected in the ovary after 18 and 36 mg/kg, respectively. The overall results provide evidence that, after intraperitoneal administration, paclitaxel concentrates in peritoneal organs; however, the intraperitoneal route does not prevent systemic drug exposure, allowing high and sustained levels of paclitaxel also in several extraperitoneal tissues.

Dissociation between in vitro cytotoxicity and in vivo cardiotoxicity of two new anthracyclines: 3'-deamino-3'-(2-methoxy-4-morpholinyl)doxorubicin and 4'-deoxy-4'-iodo-doxorubicin.

The purpose of this study was to examine the cytotoxicity and cardiotoxicity of new doxorubicin (DXR) derivatives, 3'-deamino-3'-(2-methoxy-4- morpholinyl)DXR (MRA-MT), and 4'-deoxy-4'-iodo-doxorubicin (IDXR), comparing them to doxorubicin (DXR). Both anthracycline derivatives were approximately 1.5- to 9-fold more active than DXR in inhibiting the colony-formation ability of DU145, HOS, and A2780 human cancer cell lines. Anesthetized rats given a single intravenous (i.v.) dose of DXR 10 mg/kg showed significant changes in both ECG (S alpha T segment and QRS complex widening) and hemodynamic parameters (impairment in systemic arterial dP/dtmax systolic and diastolic blood pressure), whereas animals that received MRA-MT (0.1 and 0.3 mg/kg) had no significant signs of acute cardiotoxicity. In this case the animals treated with IDXR 1.2 mg/kg showed alterations in the ECG as the animals treated with DXR. In the chronic cardiotoxicity study, some animals received MRA-MT (0.03 mg/kg i.v. once a week for 3 weeks) and others IDXR (4 mg/kg once a week for 3 weeks). They did not show any alteration in ECG and cardiac histological picture. By contrast, DXR (3 mg/kg i.v. once a week for 3 weeks) induced a severe cardiomyopathy, characterized by progressive widening of S alpha T segment, increase in T wave, and histological damage consisting of vacuolations and loss of myofibrils. These results suggest that MRA-MT and IDXR are more active in vitro and markedly less cardiotoxic in vivo than DXR.