Bortezomib overcomes MGMT-related resistance of glioblastoma cell lines to temozolomide in a schedule-dependent manner.

Development of drug resistance after standard chemotherapy for glioblastoma multiforme (GBM) with temozolomide (TMZ) is associated with poor prognosis of GBM patients and is at least partially mediated by a direct DNA repair pathway involving O6-methylguanine methyltransferase (MGMT). This enzyme is under post-translational control by a multisubunit proteolytic cellular machinery, the 26S proteasome. Inhibition of the proteasome by bortezomib (BZ), a boronic acid dipeptide already in clinical use for the treatment of myeloma, has been demonstrated to induce growth arrest and apoptosis in GBM cells. In this study we investigated the effect of sequential treatment with BZ and TMZ on cell proliferation-viability and apoptosis of the human T98G and U87 GBM cell lines. We also tested for an effect of treatment on MGMT expression and important upstream regulators of the latter, including nuclear factor kappa B (NFκB), p44/42 mitogen-activated protein kinase (MAPK), p53, signal transducer and activator of transcription 3 (STAT3) and hypoxia-inducible factor 1α (HIF-1α). The sequence of drug administration for maximal cytotoxicity favored BZ prior to TMZ in T98G cells while the opposite was the case for U87 cells. Maximal efficacy was associated with downregulation of MGMT, reduced IκBα-mediated proteasome-dependent nuclear accumulation of NFκB, attenuation of p44/42 MAPK, AKT and STAT3 activation, and stabilization of p53 and inactive HIF-1α. Collectively, these results suggest that proteasome inhibition by BZ overcomes MGMT-mediated GBM chemoresistance, with scheduling of administration being critical for obtaining the maximal tumoricidal effect of combination with TMZ.

Bortezomib reverses the proliferative and antiapoptotic effect of neuropeptides on prostate cancer cells.

OBJECTIVES: Neuropeptides are important signal initiators in advanced prostate cancer, partially acting through activation of nuclear factor kappa B. Central to nuclear factor kappa B regulation is the ubiquitin-proteasome system, pharmacological inhibition of which has been proposed as an anticancer strategy. We investigated the putative role of the proteasome inhibitor bortezomib in neuropeptides signaling effects on prostate cancer cells. METHODS: Human prostate cancer cell lines, LNCaP and PC-3, were used to examine cell proliferation, levels of proapoptotic (caspase-3, Bad) and cell cycle regulatory proteins (p53, p27, p21), as well as total and phosphorylated Akt and p44/42 mitogen-activated protein kinase proteins. Furthermore, 20S proteasome activity, subcellular localization of nuclear factor kappa B and transcription of nuclear factor kappa B target genes, interleukin-8 and vascular endothelial growth factor, were assessed. RESULTS: Neuropeptides (endothelin-1, bombesin) increased cell proliferation, whereas bortezomib decreased proliferation and induced apoptosis, an effect maintained after cotreatment with neuropeptides. Bad, p53, p21 and p27 were downregulated by neuropeptides in PC-3, and these effects were reversed with the addition of bortezomib. Neuropeptides increased proteasomal activity and nuclear factor kappa B levels in PC-3, and these effects were prevented by bortezomib. Interleukin-8 and vascular endothelial growth factor transcripts were induced after neuropeptides treatment, but downregulated by bortezomib. These results coincided with the ability of bortezomib to reduce mitogen-activated protein kinase signaling in both cell lines. CONCLUSIONS: These findings are consistent with bortezomib-mediated abrogation of neuropeptides-induced proliferative and antiapoptotic signaling. Thus, the effect of the drug on the neuropeptides axis needs to be further investigated, as neuropeptide action in prostate cancer might entail involvement of the proteasome.

Bortezomib represses HIF-1α protein expression and nuclear accumulation by inhibiting both PI3K/Akt/TOR and MAPK pathways in prostate cancer cells.

Bortezomib represents the first proteasome inhibitor (PI) with demonstrated antitumor activity in the clinical setting, particularly for treatment of hematological malignancies. At the preclinical level, its action is shown to be mediated by induction of growth arrest and apoptosis in many tumor types, including androgen-dependent (AD) and androgen-independent (AI) prostate cancer (PCa) cells. Hypoxia-inducible factor-1α (HIF-1α), which is directly involved in tumor growth, is one of the most studied and promising molecular targets for anti-cancer therapy and is often overexpressed in PCa. Bortezomib has been reported to impair tumor growth by also inhibiting HIF-1α. In this study, we investigated the effect of bortezomib on the expression, activity and localization of HIF-1α in LNCaP (AD) and PC3 (AI) PCa cells. First, we show that hypoxic upregulation of HIF-1α protein levels and activity involves both the PI3K/Akt/mTOR and p44/42 MAPK pathways. Second, bortezomib inhibits expression of HIF-1α protein under both normoxic and hypoxic conditions, represses HIF-1 transcriptional activity and attenuates the release of vascular endothelial growth factor. These effects correlate with the ability of bortezomib to cause dephosphorylation of phospho-Akt, phospho-p70S6K, and phospho-S6RP, thus inactivating a pathway known to be required for HIF-1α protein expression at the translational level. Furthermore, bortezomib also abrogates p44/42 MAPK phosphorylation, which results to reduced nuclear translocation of HIF-1α. Taken together, these results suggest that bortezomib inhibits HIF-1α protein synthesis and its nuclear targeting through suppression of PI3K/Akt/mTOR and MAPK pathways, respectively, in both AD and AI PCa cells.

Further examination of antiradical properties of Crocus sativus stigmas extract rich in crocins.

Data for antiradical properties of saffron extract and its bioactive constituents (crocins, crocetin) are limited and poorly discussed in comparison with those of extracts containing potent scavengers. Further examination was sought using the Folin-Ciocalteu (F-C) reagent and various free radical species produced in cell-free or cell model systems. Oregano and turmeric methanol extracts, rich in well established scavengers, and also crocetin, rosmarinic acid, and curcumin, representing the major types of constituents in the three studied extracts, were used as "reference". On the same weight basis, saffron extract activity was found to be rather negligible in all cell-free systems with regard to that found for reference ones. On the contrary, in the human monocyte system, saffron extracts or free crocetin were found to reduce ROS production as effectively as the phenolic antioxidants. Our findings point out that saffron extracts exhibit a remarkable intracellular antioxidant activity that cannot be revealed using assays repeatedly applied to the evaluation of phenolic-type antioxidants.

Oral calcium supplementation ambulatory blood pressure and relation to changes in intracellular ions and sodium-hydrogen exchange.

BACKGROUND: Calcium (Ca2+) supplementation has been shown paradoxically to reduce intracellular Ca2+ and induce vascular relaxation. The aim of the study was to assess 24-h blood pressure (BP) change after Ca2+ supplementation and to investigate its relation to changes in intracellular ions and the activity of the first isoform of sodium-hydrogen exchange (NHE-1) in subjects with hypertension and type 2 diabetes. METHODS: This parallel, randomized controlled, single-blinded trial, consisted of 31 patients with type 2 diabetes, and hypertension who were allocated to receive 1,500 mg of Ca2+ per day (n = 15) or no treatment (n = 16) for 8 weeks. RESULTS: In the Ca2+ group a decrease of 1.7 +/- 2.7 mm Hg (mean +/- SE) P = 0.52 for mean 24-h systolic BP (SBP) and 2.1 +/- 1.5 mm Hg, P = 0.19 for mean 24-h diastolic BP (DBP) was recorded. Whereas in the control group an increase of 1.4 +/- 2.7 mm Hg, P = 0.59 for mean 24-h SBP and 1.2 +/- 2.8 mm Hg, P = 0.83 for mean 24-h DBP was observed. Intraplatelet Ca2+ decreased whereas intraplatelet magnesium (Mg2+) and erythrocyte K+ increased in the intervention group. Change in mean 24-h SBP in the pooled group correlated with both change in intraplatelet Ca2+ (r = 0.49, P < 0.05) and NHE-1 activity (r = 0.6, P < 0.001). The contribution of intraplatelet Ca2+ was attenuated when both parameters were entered in a multivariate regression model. CONCLUSIONS: The present study shows a weak, statistically nonsignificant trend towards association of Ca2+ supplementation on 24-h BP in hypertensive subjects with type 2 diabetes. However, our results indicated an interrelation of [Ca2+]i levels and NHE-1 activity on BP in patients with hypertension and type 2 diabetes.

Prooxidant-antioxidant balance, peroxide and catalase activity in the aqueous humour and serum of patients with exfoliation syndrome or exfoliative glaucoma.

BACKGROUND: Oxidative stress plays an important role in the pathobiology of exfoliation syndrome (XFS) and exfoliative glaucoma (XFG). METHODS: We investigated the prooxidant-antioxidant balance (PAB) in aqueous humour and serum samples of 20 consecutive cases of XFS, 20 of XFG, and 20 age-matched controls, employing a recently described novel assay. The activity of catalase and the levels of (hydrogen) peroxide were also measured in these samples. RESULTS: There was no significant difference between the PAB in the aqueous humour of the XFS group (82.5 +/- 10 AU) and age-matched control patients (78.9 +/- 13.4 AU; p > 0.05). A significant shift of the PAB balance in favour of oxidants was detected in the XFG group (90.2 +/- 7.6 AU) compared with controls (p < 0.001). In the serum of patients with XFS (138.8 +/- 13.2 AU) and XFG (124.08 +/- 13.50 AU), PAB was significantly altered in favour of oxidants as compared to age-matched controls (114.9 +/- 9.91 AU); p < 0.001). Catalase activity in the aqueous from XFS (10.1 +/- 4.5 U/ml) and XFG (12.2 +/- 6 U/ml) patients was significantly lower than that measured in the normal aqueous (14.6 +/- 1.9 U/ml). Similarly, a significantly lower catalase activity was found in XFS (103 +/- 21.4 U/ml) and XFG (116 +/- 38 U/ml) serum samples compared with controls (189.6 +/- 84.3 U/ml). Finally, (hydrogen) peroxide concentration in aqueous and serum samples from patients with XFS (aqueous: 26.9 +/- 6.6 microM; serum: 41 +/- 10 microM) and XFG (aqueous: 21.7 +/- 7 microM; serum: 32 +/- 4 microM) were significantly higher than that of the controls (aqueous: 9.6 +/- 5.8 microM; serum: 24 +/- 9 microM; p < 0.001). CONCLUSIONS: These findings suggest that in XFS oxidative stress is counterbalanced in the aqueous, whereas the development of XFG is accompanied by a disruption of this balance in favour of oxidants.