Activated ClpP kills persisters and eradicates a chronic biofilm infection.

Chronic infections are difficult to treat with antibiotics but are caused primarily by drug-sensitive pathogens. Dormant persister cells that are tolerant to killing by antibiotics are responsible for this apparent paradox. Persisters are phenotypic variants of normal cells and pathways leading to dormancy are redundant, making it challenging to develop anti-persister compounds. Biofilms shield persisters from the immune system, suggesting that an antibiotic for treating a chronic infection should be able to eradicate the infection on its own. We reasoned that a compound capable of corrupting a target in dormant cells will kill persisters. The acyldepsipeptide antibiotic (ADEP4) has been shown to activate the ClpP protease, resulting in death of growing cells. Here we show that ADEP4-activated ClpP becomes a fairly nonspecific protease and kills persisters by degrading over 400 proteins, forcing cells to self-digest. Null mutants of clpP arise with high probability, but combining ADEP4 with rifampicin produced complete eradication of Staphylococcus aureus biofilms in vitro and in a mouse model of a chronic infection. Our findings indicate a general principle for killing dormant cells-activation and corruption of a target, rather than conventional inhibition. Eradication of a biofilm in an animal model by activating a protease suggests a realistic path towards developing therapies to treat chronic infections.

Valproic acid sensitizes human glioma cells for temozolomide and γ-radiation.

Temozolomide (TMZ) is given in addition to radiotherapy in glioma patients, but its interaction with the commonly prescribed antiepileptic drug valproic acid (VPA) is largely unknown. Induction of DNA demethylation by VPA could potentially induce expression of the O(6)-methylguanine-DNA-methyltransferase (MGMT) protein, causing resistance to TMZ and thereby antagonizing its effect. Therefore, this study investigates the interaction between VPA, TMZ, and γ-radiation. Two glioma cell lines were used that differ in TMZ sensitivity caused by the absence (D384) or presence (T98) of the MGMT protein. VPA was administered before (24/48 h) or after (24 h) single doses of γ-radiation; or, after 24 h, VPA treatment was accompanied by a single dose of TMZ for another 24 h. For trimodal treatment the combination of VPA and TMZ was followed by single doses of γ-radiation. In both cell lines VPA caused enhancement of the radiation response after preincubation (DMF(0.2) 1.4 and 1.5) but not after postirradiation (DMF(0.2) 1.1 and 1.0). The combination of VPA and TMZ caused enhanced cytotoxicity (DMF(0.2) 1.7) in both the TMZ-sensitive cell line (D384) and the TMZ-resistant cell line (T98). The combination of VPA and TMZ caused a significant radiation enhancement (DMF(0.2) 1.9 and 1.6) that was slightly more effective than that of VPA alone. VPA does not antagonize the cytotoxic effects of TMZ. Preincubation with VPA enhances the effect of both γ-radiation and TMZ, in both a TMZ-sensitive and a TMZ-resistant human glioma cell line. VPA combined with TMZ may lead to further enhancement of the radiation response.

Absence of the MGMT protein as well as methylation of the MGMT promoter predict the sensitivity for temozolomide.

BACKGROUND: The DNA repair protein O(6)-methylguanine-DNA methyltransferase (MGMT) can cause resistance to the alkylating drug temozolomide (TMZ). The purpose of this study was to determine the relationship between the MGMT status, determined by means of several techniques and methods, and the cytotoxic response to TMZ in 11 glioblastoma multiforme (GBM) cell lines and 5 human tumour cell lines of other origins. METHODS: Cell survival was analysed by clonogenic assay. The MGMT protein levels were assessed by western blot analysis. The MGMT promoter methylation levels were determined using methylation-specific multiplex ligation-dependent probe amplification (MS-MLPA) and quantitative real-time methylation-specific PCR (qMSP). On the basis of the results of these techniques, six GBM cell lines were selected and subjected to bisulphite sequencing. RESULTS: The MGMT protein was detected in all TMZ-resistant cell lines, whereas no MGMT protein could be detected in cell lines that were TMZ sensitive. The MS-MLPA results were able to predict TMZ sensitivity in 9 out of 16 cell lines (56%). The qMSP results matched well with TMZ sensitivity in 11 out of 12 (92%) glioma cell lines. In addition, methylation as detected by bisulphite sequencing seemed to be predictive of TMZ sensitivity in all six cell lines analysed (100%). CONCLUSION: The MGMT protein expression more than MGMT promoter methylation status predicts the response to TMZ in human tumour cell lines.

Feeding outside the forest: the importance of crop raiding and an invasive weed in the diet of gallery forest ring-tailed lemurs (Lemur catta) following a cyclone at the Beza Mahafaly special reserve, Madagascar.

In January 2005, a cyclone hit southern Madagascar, including the Beza Mahafaly Special Reserve, disrupting the flowering/fruiting cycle of Tamarindus indica, leaving Lemur catta without its major food resource during reproductive periods. We studied two adjacent groups of L. catta during the late gestation period, and both groups ventured outside the reserve to feed. The Red group (RG) fed daily on cultivated sweet potato (Ipomoea batatas) leaves in a nearby field, and both groups consumed leaves and stems of the invasive terrestrial flowering herb Mexican prickly poppy (Argemone mexicana), growing outside the reserve. The Green group (GG) spent significantly more time feeding than did RG, and more time feeding inside the forest compared to outside. The members of RG spent half of their time feeding in the crops, and nearly half of their diet consisted of easy-to-process sweet potato leaves. Additionally, RG defended and restricted GG's access to the crop territory. Of the two non-forest foods, A. mexicana leaves were higher in protein and most minerals (P, Mg, K and Na, but not Ca) and lower in fiber than sweet potato leaves, but sweet potato leaves were preferred by RG. L. catta is a markedly flexible primate with respect to diet, and switches to fallback foods from outside the forest during periods of low food availability. In the highly seasonal and unpredictable climate of southern Madagascar, such behavioral adaptations are important to the survival of this species.

Differential radiosensitizing potential of temozolomide in MGMT promoter methylated glioblastoma multiforme cell lines.

PURPOSE: To investigate the radiosensitizing potential of temozolomide (TMZ) for human glioblastoma multiforme (GBM) cell lines using single-dose and fractionated gamma-irradiation. METHODS AND MATERIALS: Three genetically characterized human GBM cell lines (AMC-3046, VU-109, and VU-122) were exposed to various single (0-6 Gy) and daily fractionated doses (2 Gy per fraction) of gamma-irradiation. Repeated TMZ doses were given before and concurrent with irradiation treatment. Immediately plated clonogenic cell-survival curves were determined for both the single-dose and the fractionated irradiation experiments. To establish the net effect of clonogenic cell survival and cell proliferation, growth curves were determined, expressed as the number of surviving cells. RESULTS: All three cell lines showed MGMT promoter methylation, lacked MGMT protein expression, and were sensitive to TMZ. The isotoxic TMZ concentrations used were in a clinically feasible range of 10 micromol/L (AMC-3046), 3 micromol/L (VU-109), and 2.5 micromol/L (VU-122). Temozolomide was able to radiosensitize two cell lines (AMC 3046 and VU-122) using single-dose irradiation. A reduction in the number of surviving cells after treatment with the combination of TMZ and fractionated irradiation was seen in all three cell lines, but only AMC 3046 showed a radiosensitizing effect. CONCLUSIONS: This study on TMZ-sensitive GBM cell lines shows that TMZ can act as a radiosensitizer and is at least additive to gamma-irradiation. Enhancement of the radiation response by TMZ seems to be independent of the epigenetically silenced MGMT gene.

Radiosensitization of human glioma cells by cyclooxygenase-2 (COX-2) inhibition: independent on COX-2 expression and dependent on the COX-2 inhibitor and sequence of administration.

PURPOSE: Patients with a malignant glioma have a very poor prognosis. Cyclooxygenase-2 (COX-2) protein is regularly upregulated in gliomas and might be a potential therapeutic target. The effects of three selective COX-2 inhibitors were studied on three human glioma cell lines. MATERIALS AND METHODS: The selective COX-2 inhibitors NS-398, Celecoxib and Meloxicam and three human glioma cell lines (D384, U251 and U87) were used. Cell growth was assessed by a proliferation assay, the interaction with radiation (0 - 6 Gy) was studied using the clonogenic assay and cell cycle distribution was determined by FACS (fluorescence-activated cell sorting) analysis. RESULTS: All COX-2 inhibitors reduced proliferation of the glioma cell lines irrespective of their COX-2 expression level. Incubation with 200 microM NS-398 24 h before radiation enhanced radiation-induced cell death of D384 cells and 750 microM Meloxicam resulted in radiosensitization of D384 and U87 cells. No radiosensitization was observed with COX-2 inhibitor administration after radiotherapy. Treatment of D384 with NS-398 (200 microM) or Celecoxib (50 microM) and U87 with NS-398 (200 microM) after radiation resulted even in radioprotection. CONCLUSIONS: Effectiveness of COX-2 inhibitors on cell proliferation and radio-enhancement was independent of COX-2 protein expression. The sequence of COX-2 inhibitor addition and irradiation is very important.

The importance of immunohistochemical expression of EGFr in squamous cell carcinoma of the oral cavity treated with surgery and postoperative radiotherapy.

PURPOSE: The aim of this study was to investigate the prognostic significance of epidermal growth factor (EGFr) expression in oral cavity squamous cell carcinoma (OCSCC) treated with curative surgery and postoperative radiotherapy. METHODS AND MATERIALS: This retrospective study included 165 OCSCC patients. The expression of EGFr was assessed on paraffin-embedded tissue of the primary tumor by immunohistochemistry using a monoclonal antibody directed against EGFr. Intensity of the EGFr expression was scored by two authors blinded for the clinical outcome. RESULTS: In the univariate analysis, locoregional control at 3 years (LRC) in the EGFr-negative cases was 69% compared with 77% in the EGFr-positive cases (p = 0.22). In the multivariate analysis for local control, a significant interaction was found between EGFr and overall treatment time of radiation (OTT). After stratification for EGFr expression, the OTT was of no importance in the EGFr-negative cases, whereas a significant difference in LRC was found in the EGFr-positive cases, in which the LRC after 3 years was 69% and 94% in case of an OTT of 0-42 days and >42 days, respectively (p = 0.009; hazard ratio = 3.42; 95% confidence interval, 1.28-8.96). No significant association was found between EGFr expression and overall survival. CONCLUSIONS: In the present study, no association was found between EGFr expression and outcome regarding locoregional control and overall survival. However, the results of the present study suggest that patients with squamous cell carcinoma of the oral cavity with high EGFr expression benefit more from a reduction of the overall treatment time of postoperative radiation than those with low EGFr expression.

Genetic profiling of a distant second glioblastoma multiforme after radiotherapy: Recurrence or second primary tumor?

OBJECT: In nearly all patients with glioblastoma multiforme (GBM) a local recurrence develops within a short period of time. In this paper the authors describe two patients in whom a second GBM developed after a relatively long time interval at a site remote from the primary tumor. The genetic profiles of the tumors were compared to discriminate between distant recurrence and a second primary tumor. METHODS: Both patients harboring a supratentorial GBM were treated with surgery and local high-dose radiotherapy. Local control of the disease at the primary tumor site was achieved. Within 2 years, a second GBM developed in both patients, not only outside the previously irradiated target areas but infratentorially in one patient and in the opposite hemisphere in the other. The tumors were examined for the presence of several genetic alterations that are frequently found in GBMs--a loss of heterozygosity at chromosome regions 1p36, 10pl5, 19q13, and 22q13, and at the CDKN2A, PTEN, DMBT1, and TP53 gene regions; a TP53 mutation; and EGFR amplification. In the first patient, genetic profiling revealed that the primary tumor had an allelic imbalance for markers in several chromosome regions for which the second tumor displayed a complete loss. In the second patient, genetic profiling demonstrated the presence of genetic changes in the second tumor that were identical with and additional to those found in the primary tumor. CONCLUSIONS: Based on the similarities between the genetic profiles of the primary and the second tumors in these patients, the authors decided that in each case the second distant GBM was a distant recurrence rather than a second independent primary tumor.

Study of vesicle leakage induced by melittin.

The leakage induced by melittin, a membrane-perturbing amphipathic peptide, from large unilamellar 1-palmitoyl-2-oleoylphosphatidylcholine (POPC) vesicles was studied using calcein as fluorescent marker. The extent of leakage has been found to be regulated by the melittin/lipid molar ratio. Melittin leads to the complete release of trapped calcein from some vesicles. This all-or-none mechanism leads to the co-existence of two different vesicle populations: the 'empty' and the intact one. Intervesicular migration of melittin was not observed. The results reveal a specific targeting of the lysed vesicles by melittin. The presence of negatively charged lipids (unprotonated palmitic acid or 1-palmitoyl-2-oleoylphosphatidylglycerol) in the neutral POPC matrix inhibits the lytic power of melittin; this inhibition increases with increasing surface charge density. It is proposed that the anchorage of the peptide on the charged surface prevents the formation of defects allowing leakage. A statistical model based on a random distribution of the peptide molecules on the vesicles is proposed to describe the release induced by melittin. It is proposed that about 250 melittin molecules per vesicle are required to affect the bilayer permeability and to empty a vesicle of its content. This large number suggests that leakage is more likely due to collective membrane perturbation by the peptide rather than to the formation of a well-defined pore.

Nisin promotes the formation of non-lamellar inverted phases in unsaturated phosphatidylethanolamines.

Nisin, a peptide used as a food preservative, is shown, by 31P-nuclear magnetic resonance and infrared spectroscopy, to perturb the structure of membranes formed of unsaturated phosphatidylethanolamine (PE) and to induce the formation of inverted non-lamellar phases. In the case of dioleoyl-sn-glycero-3-phosphatidylethanolamine (DOPE), nisin promotes the formation of inverted hexagonal phase. Similarly, the peptide induces the formation of an isotropic phase, most likely a cubic phase, with 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphatidylethanolamine (POPE). It is proposed that the insertion of the peptide in the bilayer shifts the amphiphilic balance by increasing the hydrophobic contribution and is at the origin of the changes in the polymorphic propensities of PE. This is supported by the fact that the presence of cholesterol in the PE bilayer inhibits the power of nisin to perturb the membrane structure, most likely because the peptide insertion is difficult in the fluid ordered phase. This finding provides insight into possible antibacterial mechanisms of nisin.

Structural characterization of free and membrane-bound nisin by infrared spectroscopy.

This study reports two new trends about nisin affinity for lipid membranes. First, there is a very strong dependence of nisin binding on the membrane surface charge. As illustrated in this work, the binding of nisin is much greater for phosphatidylglycerol (PG) than for phosphatidylcholine (PC) membranes. This can be rationalized by electrostatic attraction between the positively charged peptide and the negatively charged PG. Second, the affinity of nisin shows a very weak dependence on the lipid phase, the binding to fluid or gel phase membranes being nearly equivalent. Therefore, our results suggest that nisin behaves as an extrinsic peptide. This work also presents the first piece of information relative to the structure of membrane-bound nisin. The Amide I band of the peptide is different for free nisin in water and for membrane-bound nisin. By analyzing this region using self-deconvolution and band fitting, and by comparing with results obtained from nisin dissolved in various H2O/trifluoroethanol mixtures, it can be inferred that the binding of nisin to phospholipid membranes leads to an increased proportion of beta-turns.

Effect of cholesterol on the polymorphism of dipalmitoylphosphatidylcholine/melittin complexes: an NMR study.

In order to get insights into the effects of cholesterol on protein activity, the lytic power of melittin on 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC)/cholesterol mixtures was studied using solid-state deuterium and phosphorus-31 nuclear magnetic resonance spectroscopy (2H and 31P-NMR). After incubation, melittin disrupts pure DPPC vesicles, leading to the formation of small lipid/peptide complexes below the phase transition temperature (Tm), whereas large bilayer assemblies are reformed above Tm; the transition between these two species is thermally reversible. This study reveals that cholesterol modifies this thermal behavior and that this modulation of the lytic power of melittin is indirect, since it is essentially related to the original effect of the sterol on the thermotropism of pure lipid bilayers. It is known that melittin does not lyse gel phase DPPC bilayers spontaneously. Our study shows that the addition of large amounts of sterol (30 mol%) does not promote the spontaneous lysis at 26 degrees C, despite the increased fluidity of the lipid system. The lysis takes place around 32 degrees C, regardless of the cholesterol concentration. This study also shows that high concentrations of cholesterol (> or = 30%) in DPPC bilayer inhibit the lysis. It is proposed that the tight lipid packing due to high cholesterol concentrations prevents the penetration of melittin into the bilayer. When melittin interacts with cholesterol-rich bilayers (30 mol%), the lysis is only partial, and leads to the formation of small cholesterol-depleted particles. Finally, DPPC which bears deuteriated acyl chains was used to determine the influence of melittin on the orientational order of the lipid chains in the large assemblies. The quadrupolar splittings obtained in the presence of melittin are not considerably different than those obtained in the absence of melittin.

A restatement of melittin-induced effects on the thermotropism of zwitterionic phospholipids.

Perturbations induced by melittin on the thermotropism of dimyristoyl-, dipalmitoyl-, distearoylphosphatidylcholine and natural sphingomyelin are investigated and rationalized from data obtained by fluorescence polarization, differential scanning calorimetry and Raman spectroscopy. Depending on the technique and/or experimental conditions used, the observed effects differ at the same lipid to protein molar ratio, due to partial binding of melittin. The binding is more efficient for tetrameric than for monomeric melittin, but in both cases its affinity is weaker for phosphatidylcholine dispersions in the gel phase than for sonicated vesicles. For temperatures T greater than or equal to Tm efficient binding occurs whatever the initial state of the lipids is. One can summarize the effects induced by melittin on the transition temperature as follows: No upward shift is observed on synthetic phosphatidylcholines when lipid degradation is avoided. This is achieved by using highly purified melittin, phospholipase inhibitors, and/or non-hydrolysable lipids. Melittin monomer does not change Tm. When melittin tetramer is stabilized, it decreases Tm by 10-15 deg. C. The transition broadens, and is finally abolished for Ri less than or equal to 2. Very similar results are found for natural sphingomyelin. Fluorescence polarization indicates similar changes in order and dynamics of the acyl chains for all lipid studied. For T less than or equal to Tm, fluorescence and Raman show that melittin decreases the amount of CH2 groups in 'trans' conformation and the intermolecular order of the chains. According to fluorescence data, there is an increase of the rigid-body orientational order at T greater than or equal to Tm, while from Raman the positional intermolecular order decreases without significant change in the CH2 groups 'trans'/'gauche' ratio.

Differential scanning calorimetry and (2)H nuclear magnetic resonance and Fourier transform infrared spectroscopy studies of the effects of transmembrane alpha-helical peptides on the organization of phosphatidylcholine bilayers.

We have studied the effects of the incorporation of the alpha-helical transmembrane peptides Ac-K(2)-L(24)-K(2)-amide (L(24)) and Ac-K(2)-(L-A)(12)-K(2)-amide ((LA)(12)) on the thermotropic phase behavior of 1,2-dipalmitoyl-d(62)-sn-glycero-3-phosphocholine (DPPC-d(62)) and 1-palmitoyl-d(31)-2-oleoyl-sn-glycero-3-phosphocholine (POPC-d(31)) lipid bilayer model membranes by differential scanning calorimetry (DSC) and the conformational and orientational order of the phospholipid chains by Fourier transform infrared (FTIR) spectroscopy and (2)H nuclear magnetic resonance ((2)H-NMR) spectroscopy, respectively. Our DSC and FTIR spectroscopic studies indicate that the peptides L(24) and (LA)(12) both decrease the temperature and enthalpy of the gel/liquid-crystalline phase transition of DPPC-d(62) bilayers, with (LA)(12) having the greater effect in this regard. An examination of the frequencies of the CH(2) and CD(2) symmetric stretching bands of the infrared spectra of liquid-crystalline states of the peptide-free and peptide-containing DPPC-d(62) and POPC-d(31) samples, and a comparison with the orientational order as measured by (2)H-NMR spectroscopy as well as with the chain order as measured by electron spin resonance spectroscopy, lead us to conclude that the CH(2) (or CD(2)) stretching frequencies of lipid hydrocarbon chains are not a reliable measure of chain conformational order in lipid bilayers containing significant amounts of peptides or other lipophilic inclusions. In contrast, the results of our (2)H-NMR spectroscopic studies present a consistent picture in which both L(24) and (LA)(12) increased in a similar way the time-averaged orientational order of the lipid chains of their liquid-crystalline lipid bilayer hosts. The comparison of the effects L(24) and (LA)(12) on phosphatidylcholine bilayers indicates that the gel-to-liquid-crystalline phase transition appears to be more sensitive to small changes in transmembrane peptide surface topology than hydrocarbon carbon chain orientational order in the liquid-crystalline state.

Perturbation of binary phospholipid mixtures by melittin: a fluorescence and raman spectroscopy study.

The effect of melittin on different binary mixtures of phospholipids has been studied by polarization of DPH fluorescence in order to determine if melittin can induce phase separation. Since the interaction between lipids and melittin is sensitive to both electrostatic and hydrophobic forces, we have studied the effect of the acyl chain length and of the polar head group of the lipids. In spite of the difference of the chain length between dipalmitoylphosphatidylcholine (DPPC) and distearoylphosphatidylcholine (DSPC), no phase separation occurs in an equimolar mixture of these lipids in presence of melittin. However, when the charged lipid dipalmitoylphosphatidylglycerol (DPPG) is mixed with either DPPC or DSPC, the addition of melittin leads to phase separation. The DSPC/DPPG/melittin system, which shows a very complex thermotropism, has also been studied by Raman spectroscopy using DPPG with deuteriated chains in order to monitor each lipid independently. The results suggest that the higher affinity of melittin for DPPG leads to a partial phase separation. We propose the formation of DPPG-rich domains perturbed by melittin and peptide-free regions enriched in DSPC triggered by the head group charge and chain-length differences.

Singlet molecular oxygen causes loss of biological activity in plasmid and bacteriophage DNA and induces single-strand breaks.

Damage of plasmid and bacteriophage DNA inflicted by singlet molecular oxygen (1O2) includes loss of the biological activity measured as transforming capacity in E. coli and single-strand break formation. Three different sources of 1O2 were employed: (i) photosensitization with Rose bengal immobilized on a glass plate physically separated from the solution; (ii) thermal decomposition of the water-soluble endoperoxide 3,3'-(1,4-naphthylidene) dipropionate (NDPO2); and (iii) microwave discharge. Loss of transforming activity was documented after exposing bacteriophage M13 DNA to 1O2 generated by photosensitization employing immobilized Rose bengal, and with bacteriophage luminal diameter X174 DNA, using the thermodissociable endoperoxide (NDPO2) as a source of 1O2. These findings are in agreement with experiments in which plasmid DNA pBR322 was exposed to a gas stream of 1O2 generated by microwave discharge. The effects of 1O2 quenchers and of 2H2O indicate 1O2 to be the species responsible. Strand-break formation in pBR322 and luminal diameter X174, measured as an increase of the open circular form at the expense of the closed circular supercoiled form, was observed without alkaline treatment after exposing the DNA to 1O2, using either agarose gel electrophoresis or sucrose gradient separation. The effect of quenchers and 2H2O indicate the involvement of 1O2 in DNA damage. We conclude that singlet oxygen can cause loss of biological activity and DNA strand breakage.

Expression of cyclooxygenase-2 and epidermal growth factor receptor in primary and recurrent glioblastoma multiforme.

PURPOSE: To investigate the pattern and level of cyclooxygenase-2 (COX-2) expression in a series of high grade primary and recurrent glioblastoma multiforme (GBM) and correlation with time to recurrence and patients' survival following therapy. The relationship between COX-2 and epidermal growth factor receptor (EGFR) immunoreactivities was evaluated. MATERIALS AND METHODS: Specimens of 14 primary and 14 recurrent GBMs (eight pairs) following surgery and full course radiation therapy were processed for immunostaining on COX-2 and EGFR. Tumor cell positivity was semi-quantitatively scored. COX-2 scores of the primary tumor and recurrence were correlated with the time to radiological tumor progression and patients' survival. RESULTS: COX-2 positive tumor cells were disseminated throughout the tumor parenchyma. The intensity and pattern of COX-2 expression were heterogeneous, with predominant expression in areas surrounding tumor necrosis. Scoring of COX-2 positivity revealed values between 1 and 80% of the cells. Primary GBMs with COX-2 expression levels between 25% and 70% of the tumor cells showed a shorter time to radiological recurrence than GBMs with <10% COX-2 positive tumor cells (respectively, 219 +/- 50 and 382 +/- 77 days). No correlation was found between the COX-2 expression in the primary tumor and patients' survival (r (s) = -0.073) following therapy. No correlation was found either between COX-2 and EGFR immunoreactivity. CONCLUSIONS: Immunohistochemical expression of COX-2 in GBM showed large variation. Hence, determination of COX-2 expression in tumor specimen for each individual might be relevant for selection of those patients, who could benefit from adjuvant therapy with selective COX-2 inhibitors.

Targeting anti-apoptotic Bcl-2 by AT-101 to increase radiation efficacy: data from in vitro and clinical pharmacokinetic studies in head and neck cancer.

BACKGROUND: Pro-survival Bcl-2 family members can promote cancer development and contribute to treatment resistance. Head and neck squamous cell carcinoma (HNSCC) is frequently characterized by overexpression of anti-apoptotic Bcl-2 family members. Increased levels of these anti-apoptotic proteins have been associated with radio- and chemoresistance and poor clinical outcome. Inhibition of anti-apoptotic Bcl-2 family members therefore represents an appealing strategy to overcome resistance to anti-cancer therapies. The aim of this study was to evaluate combined effects of radiation and the pan-Bcl-2 inhibitor AT-101 in HNSCC in vitro. In addition, we determined human plasma levels of AT-101 obtained from a phase I/II trial, and compared these with the effective in vitro concentrations to substantiate therapeutic opportunities. METHODS: We examined the effect of AT-101, radiation and the combination on apoptosis induction and clonogenic survival in two HNSCC cell lines that express the target proteins. Apoptosis was assessed by bis-benzimide staining to detect morphological nuclear changes and/or by propidium iodide staining and flow-cytometry analysis to quantify sub-diploid apoptotic nuclei. The type of interaction between AT-101 and radiation was evaluated by calculating the Combination Index (CI) and by performing isobolographic analysis. For the pharmacokinetic analysis, plasma AT-101 levels were measured by HPLC in blood samples collected from patients enrolled in our clinical phase I/II study. These patients with locally advanced HNSCC were treated with standard cisplatin-based chemoradiotherapy and received dose-escalating oral AT-101 in a 2-weeks daily schedule every 3 weeks. RESULTS: In vitro results showed that AT-101 enhances radiation-induced apoptosis with CI's below 1.0, indicating synergy. This effect was sequence-dependent. Clonogenic survival assays demonstrated a radiosensitizing effect with a DEF37 of 1.3 at sub-apoptotic concentrations of AT-101. Pharmacokinetic analysis of patient blood samples taken between 30 min and 24 h after intake of AT-101 showed a dose-dependent increase in plasma concentration with peak levels up to 300-700 ng/ml between 1.5 and 2.5 h after intake. CONCLUSION: AT-101 is a competent enhancer of radiation-induced apoptosis in HNSCC in vitro. In addition, in vitro radiosensitization was observed at clinically attainable plasma levels. These finding support further evaluation of the combination of AT-101 with radiation in Bcl-2-overexpressing tumors.

L-Dopa stimulates expression of the antioxidant enzyme NAD(P)H:quinone oxidoreductase (NQO) in cultured astroglial cells.

The autooxidation of L-Dopa, a catecholamine used in the symptomatic treatment of Parkinson's disease, generally yields reactive oxygen species and neurotoxic quinones. NAD(P)H:quinone oxidoreductase (NQO) is a flavoenzyme that is implicated in the detoxication of quinones, including those formed during L-Dopa autooxidation. Through the action of this enzyme, deleterious redox-labile quinones are turned into less toxic and more stable hydroquinones that are amenable to further detoxication and/or cellular excretion. In the present study, using primary rat astrocytes and C6 astroglioma as a model to evaluate the neuroprotective response of astroglial cells upon exposure to L-Dopa, we demonstrate that this compound, or more correctly its quinone (auto)oxidation products, up-regulates astroglial NQO in a time- and concentration-dependent way as assessed at the level of mRNA expression, protein level, and enzymatic activity. Moreover, under similar conditions cellular glutathione content was enhanced. It is concluded that, similar to glutathione, the oxidative stress limiting NQO is likely to contribute to the capacity of astroglial cells to protect dopaminergic neurons against L-Dopa, and, hence, may be considered as a potential target for the development of neuroprotective strategies for Parkinson's disease.

Modulation by D,L-buthionine-S,R-sulphoximine of etoposide cytotoxicity on human non-small cell lung, ovarian and breast carcinoma cell lines.

Treatment with 25 mumol/l D,L-buthionine-S,R-sulphoximine (BSO) for at least 24 h depleted glutathione (GSH) in human non-small cell lung (SW-1573), ovarian (A2780) and breast carcinoma (MCF-7) cell lines to about 20% of control, and was accompanied by a 2-fold potentiation of the cytotoxicity of etoposide, doxorubicin and cisplatin. Cellular etoposide, but not doxorubicin or cisplatin, concentrations were increased 2-fold due to decreased efflux. This occurred independently of the presence of BSO during 1 h of etoposide exposure, but required prolonged exposure to BSO (at least 24 h). Energy depletion as well as cotreatment, but not pretreatment, of the cells with daunomycin, doxorubicin, vinblastine or vincristine increased cellular etoposide accumulation. Treatment of control cells with verapamil caused similar changes in etoposide cytotoxicity and cellular pharmacokinetics as GSH depletion, but did not further increase etoposide cytotoxicity and accumulation in GSH-depleted cells. Etoposide efflux may have been inhibited, not because of (competitive) inhibition by BSO or disturbance of the energy required for this process, but probably because of plasma membrane alterations.