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The aim of this study is to develop co-encapsulation of quercetin (QCT) and superparamagnetic iron oxide nanoparticles (SPIONs) into methoxy-poly(ethylene glycol)-b-oligo(ɛ-caprolactone), mPEG750-b-OCL-Bz micelles (QCT-SPION-loaded micelles) for inhibition of hepatitis B virus-transfected hepatocellular carcinoma (HepG2.2.15) cell growth. QCT-SPION-loaded micelles were prepared using film hydration method. They were spherical in shape with an average size of 22-55 nm. The best QCT-SPION-loaded micelles showed entrapment efficiency and loading capacity of QCT at 70% and 3.5%, respectively, and of SPIONs at 15% and 0.8%, respectively. Transverse (T2) relaxivity of SPIONs was 137 mM-1s-1. SPION clusters present inside the core of QCT-SPION-loaded micelles increased T2 relaxivity value (246 mM-1s-1) indicating the good magnetic resonance imaging sensitivity of QCT-SPION-loaded micelles in comparison with SPIONs. QCT-SPION-loaded micelles could be taken up by HepG2.2.15 cells and showed higher cytotoxicity than QCT. Furthermore, these cells were arrested by QCT-SPION-loaded micelles at the G0/G1 phase of cell cycle. QCT-SPION-loaded micelles accumulated in the vicinity of Neodymium Iron Boron (NdFeB) magnetic disc, resulting in the potent inhibition of cancer cell growth at the strong magnetic field strength. In conclusion, mPEG750-b-OCL-Bz micelles are a promising multi-functional vehicle for co-delivery of QCT and SPIONs for disease monitoring and therapies of hepatocellular carcinoma.

We investigated the possibility to grow molecularly porous amino-silane layers on glass-like substrates. The goal of this work is to show that it is possible to substantially increase the sensitivity of a fluorescence sensor by adjusting the functionalization strategy. Two methods are studied, one using APTMS only and another one using both APTMS and APDMS. We show that, using the second method, sensor sensitivity is improved by a factor of about 5. In order to demonstrate this, we applied the technique to the grafting of fluorescein in order to build a fluorescence pH sensor.

Keloids are pathologic scars defined as dermal fibrotic tumors resulting from a disturbance of skin wound healing process. Treatments against keloids are multiple, sometimes empirical and none of them really provides an effective tool for physicians. The lack of effective treatments is correlated with the poor understanding of keloid pathogenesis. To fill this gap, researchers need strong models mimicking keloids as closely as possible. The objective of this study was to establish in vitro a new reconstructed keloid model (RKM), by combining fibroblasts extracted from the three major area of a keloid (center, periphery, non-lesional) in a three-dimensional biomaterial. To this aim, fibroblasts of three keloid locations were extracted and characterized, and then integrated in a hydrated collagen gel matrix during a three-step procedure. The heterogeneity of fibroblasts was assessed according to their proliferative and remodeling capacities. RKMs were further visualized and characterized by both light and scanning electron microscopy. This reconstructed keloid model should be very useful for investigating keloid fibroblasts function in conditions mimicking in vivo situation. Moreover, RKM should also be a suitable model for either drug study and discovery or innovative approaches using medical devices both during cancer and cancer-like disease investigation.

International audience; Organic functions of the human body are related to biological constants. Variations of these constants, among them pH, induce pathological troubles. The general goal of our work is to fabricate a fluorescent pH sensor at the end of an optical fiber for in vivo pH measurements. One difficulty using fluorescence indicators is the need to perform an accurate calibration. In this communication, we present methods used to simplify and potentially avoid calibration procedures of fluorescence indicators. The first method concerns the simplification of calibration procedures making them independent of the indicator’s concentration, path length and equipment used. The second method concerns modelling the fluorescence emission of the molecules as a function of pH only. This model is used to fit the exact shape of C-SNARF-1 fluorescence spectra obtained at any pH. Subsequently, the pH of a solution can be computed with an accuracy of 0.1 pH unit without the calibration procedure employed up to now. These methods constitute the first steps toward calibration free pH measurements. They can be applied to any fluorescent indicator exhibiting a dual emission peak. As a conclusion, this is the first time that fluorescence properties of C-SNARF-1 are fully mathematically described.

This research was designed to visualize cellular response – in particular the generation of intracellular ROS – to stimulation by polycyclic aromatic hydrocarbon (PAH). We used chloroauric acid (HAuCl4) as a molecular probe for visualizing intracellular ROS generated by the stimulation of benzo[a]pyrene (BaP). The chloroauric acid undergoes a spontaneous reduction reaction, assisted by the intracellular ROS, into gold nanoclusters (AuNCs). As a result, we can visualize the ROS via optical imaging technique. According to MTT assay, the chloroauric acid exhibited good biocompatibility. The AuNCs produced in the cells were approximately 2–3 nm in diameter with a green fluorescent property. Cellular imaging showed that BaP induced the formation of AuNCs within the cells, leading to a high relative cellular fluorescent intensity with a considerable extent of scatter light. Thus, this probe is an efficient molecular imaging probe for visualizing intracellular ROS.

International audience; The goal of this work concerns a new method to substantially increase the sensitivity of a fluorescence pH sensor. The method is based on a double silanization method. Two methods are compared: one using APTMS (3-Aminopropyl)trimethoxysilane) only and another one using both APTMS and APDMS (3-Aminopropyl)dimethoxymethylsilane). Using the second method, sensor’s sensitivity is improved by more than 500 %.

Physiological responses such as chronic inflammation and angiogenesis could be used as biomarkers for early detection of cancer with noninvasive imaging modalities. The present study reports the application of magnetic resonance imaging instrument to image the binding of ferrioxamine with hemin that allows visualizing the chronic inflammation foci of lung tissue of immunocompromised rats xenografted using small cell lung carcinoma. A low concentration of ferrioxamine (0.05±0.02 μM·kg−1of rat weight) deposited on tissue outside the vasculature was found to diffuse across the capillary walls to the interstitial space and inflammation foci, which provided a clear enhancement of T1-weighted gradient-echo sequence images. Ferrioxamine imaging allowed the determination of inflammatory sites and their localization in 3D fat-suppressed maximum intensity projections. The smallest dimension of foci that can be clearly determined is about 0.1 mm3. In concomitant to thein vivoimaging, analysis of histological tissue section showed the development of inflammatory sites. This study provides evidence that medical imaging instrument such as MRI scanner allows researchers to correlate images taken with MRI with those using high-resolution microscopy. Moreover, ferrioxamine is a useful molecular probe for determining chronic inflammation particularly at the very early stages of cancer.

In this study, quercetin (QCT), a flavonoid with high anticancer potential, was loaded into polymeric micelles of PEG-OCL (poly(ethylene glycol)-b-oligo(ε-caprolactone)) with naphthyl or benzyl end groups in order to increase its aqueous solubility. The cytostatic activity of the QCT-loaded micelles toward different human cancer cell lines and normal cells was investigated. The results showed that the solubility of QCT entrapped in mPEG750-b-OCL micelles was substantially increased up to 1 mg/ml, which is approximately 110 times higher than that of its solubility in water (9 μg/ml). The average particle size of QCT-loaded micelles ranged from 14 to 19 nm. The QCT loading capacity of the polymeric micelles with naphthyl groups was higher than that with benzyl groups (10% and 6%, respectively). QCT-loaded, benzyl- and naphthyl-modified micelles effectively inhibited the growth of both sensitive and resistance cancer cells (human erythromyelogenous leukemia cells (K562) and small lung carcinoma cells (GLC4)). However, the benzyl-modified micelles have a good cytocompatibility (in the concentration range investigated (up to 100 μg/ml), they are well tolerated by living cells), whereas their naphthyl counterparts showed some cytotoxicity at higher concentrations (60-100 μg/ml). Flow cytometry demonstrated that the mechanism underlying the growth inhibitory effect of QCT in its free form was inducing cell cycle arrest at the G2/M phase. Benzyl-modified micelles loaded with QCT also exhibited this cycle arresting the effect of cancer cells. In conclusion, this paper shows the enhancement of solubility and cell cycle arrest of QCT loaded into micelles composed of mPEG750-b-OCL modified with benzyl end groups. These micelles are therefore considered to be an attractive vehicle for the (targeted) delivery of QCT to tumors.

Problem statement: We have previously analyzed the interaction of BSA with flavonoids by using FRET. In this study, the role of BSA on increasing in solubility and on carrying the quercetin derivatives thus enhanced their anticancer efficacy against drug-sensitive and drug-resistant cells were conducted. Approach: The macroscopic (KD) and microscopic (kd) binding constant of the complexation and the cellular partition of molecules were analyzed using FRET and HPLC method, respectively. Results: The KD values reflex the stability of complexes was in the order of rutin > quercetrin > quercetin. BSA was a suitable carrier of quercetin (KD = 1.68×105 M−1) which spontaneously release the molecule into solutions and cells. The substitution of rhamnoside (KD = 1.37×105 M−1) and rutinoside (KD = 5.0×104 M−1) at C3 yielded an increase in stability of the complexes. Rutin was tightly bound to BSA resulting in the changes in mode of action. Conclusion: The macroscopic binding constant was directly influenced on the cellular uptake of molecules and the suitable range of binding constant was KD≥105 M−1 by which the carrier can be useful for increasing the solubility of drug and also spontaneously release the drug into the solution and cells.

Problem statement: Cellular drug resistance to anticancer agents is major obstacle in cancer chemotherapy and the mechanisms by which these MDR cells possess for protecting themselves to survive prolonged exposure to cytotoxic agents still debating. The study aimed to clarify the role of P-glycoprotein (Pgp) and enhanced drug sequestration in lysosomes to confer the multidrug resistance K562 cells with varied degree of Pgp expression. Approach: Erythromyelogenous leukemic K562 and its corresponding Pgp-over expression K562/adr (RF= 26.5) and K562/10000 (RF = 39.6) cells were used. The transport of intrinsic fluorescence molecules including acridine orange and pirarubicin across plasma membrane of living cells was performed by using spectrofluorometric and flow cytometric analysis. Results: Pirarubicin passively diffused through the plasma membrane of K562, K562/adr and K562/10000 cells with the same values of k+ = 3.4±0.3 pL. s-1.cell-1. Similar results were found for acridine orange, which passively diffused through plasma membrane of these cell lines about 30-fold faster than pirarubicin. The mean rate of Pgp-mediated efflux coefficient (ka) of pirarubicin was equal to 2.6 ± 0.9 pL.s-1.cell-1 for K562/adr and 4.7 ± 1.0 pL.s-1.cell-1 for K562/10000 cells. The Pgp-mediated efflux of acridine orange could not be determined for K562/adr cells while an enhancement of exocytosis in K562/10000 cells was characterized. The acridine orange exhibited antiproliferative activity and IC50 for K562, K562/adr and K562/10000 cells was 447±40, 715±19 and 1,719±258 nM, respectively. Cytotoxicity of acridine orange was increased by 2-fold in the presence of and 25 nM monensin. Conclusion: The results clearly demonstrated for the first time that by using the same methods and cell lines. The predominant cellular defense mechanism determined in multidrug resistant cells depends upon the nature of molecular probes used. As molecular probe, pirarubicin clearly showed that the Pgp-mediated efflux of drug play as predominant mechanism while AO clearly demonstrated the role of drug sequestration in lysosomes following an enhance exocytosis in both MDR sublines.

Multidrug resistance was an obstacle in cancer chemotherapy because the cells decreased their intracellular drug accumulation by energy-dependent compounds efflux pumps such as P-glycoprotein (P-gp). This study observed some iodinated radiographic contrast media, diatrizoate, iopromide and iotrolan affected the cellular energetic state and the kinetics of P-gp in drug-sensitive K562 and drug resistant K562/adr cell lines using spectrophotometer and spectrofluorometer. By colorimetric MTT assay, it was found that contrast media (0-3500 μM) had no effect on both K562 and K562/adr cell viabilities, but in co-treatment with daunorubicin (DNR), diatrizoate decreased cell viability in K562/adr cells by decreasing ICso of DNR from 610.7 ±74.5 nM to 360±108.9 nM. The change in cellular energetic state was studied using rhodamine B as a probe to estimate mitochondrial membrane potential (ΔΨm). The results showed that 3500 μM diatrizoate decreased ΔΨm from 162.2±0.3 mV to 86.9±9.9 mV in K562/adr cells. The kinetics of P-gp-mediated efflux of DNR could be reduced by diatrizoate from 0 (no inhibition) to 0.65±0.11. This inhibition could be partially prevented in co-incubation with 20 nM concanamycin A or 10 μM cytochalasin B. Among the three molecules, diatrizoate showed the best efficiency. It could be proposed for further studies that diatrizoate could be used as MDR identification or MDR imaging and also acted as MDR sensitizing agent in cancer treatments.

Problem statement: Plant derived fungicides are now being subjects of many research groups. These secondary metabolites have enormous potential to inspire and influence modern agrochemical research. The study aimed to investigate the antifungal activity and their potential use as fungicides in the agriculture of crude extracts and purified compounds derived from plants used in traditional medicines. Approach: Four medicinal plants including A. galanga, C. longa, B. pandurata and C. odorata were selected and percolated with hexane, ethyl acetate, acetone or methanol. The extracts were purified and elucidated their chemical structures. Disc mycelial growth inhibition was applied in order to determine their anti P. capsici activity and the field study was performed to determine their potential use in controlling fungal infection in chili plants compared with commercial fungicides such as captan and bio-control Trichoderma virens. Results: All crude extract inhibited mycelial growth of the fungus performed with similar efficacy. ED90 was equal to 300 ppm. Among plants studied B. pandurata was the most potent against P. capsici. The proposed active ingredients were pinostrobin and pinocembrin. In the field study, pinocembrin mediated the same anti P. capsici activity as captan. B. pandurata can protect chili from infection, thus increasing crop yield of chili comparable to Trichoderma virens. Conclusion: The results clearly showed that the extracts of the four plants studied could be considered as potential sources of novel fungicides. Particularly, B. pandurata has a very high potential as raw material for developing the antifungal molecule of non-petrochemical, naturally eco-friendly, easily obtainable and not toxic to human beings and environment, at least for use in chili growing.

The objectives of this study were to investigate the behavior of flavonoids in an aqueous physiological buffer and to determine the structural and functional group substitution which is responsible for their anticancer action. The de- protonated anionic form of 7 flavonoids can easily be determined using spectrophotometry, and owing to its charged state, is highly soluble in aqueous physiological buffer and is not prone to aggregation. The protonated form of these 7 flavon- oids is much less soluble and tends to aggregate following precipitation. For all flavonoids studied except catechin and 5,5� -dihydroxy-6,7,3� ,4� -tetramethoxyflavone, it was possible to determine the rates of deprotonation; pKa value of eri- odictyol, apigenin, kaempferol, quercetin, WP 279, and WP 283 was equal to 7.00, 8.72, 7.86, 8.30, 7.70 and 9.90, respec- tively. The methoxyl group substitutions in place of hydrogen atoms and/or hydroxyl groups at various positions of car- bon atoms in ring A, B and C particularly WP 283 resulted in an increase in the solubility, lipophilicity, and specifically its anticancer efficacy (by 60-fold). The neutral forms of flavonoids are predominantly active molecules and the active sites responsible for anticancer activity are found in ring A and C, especially C4=O, C5-OH and C2=C3.

This article examines a non-invasive spectrofluorometric method using the 2′,7′-dichlorofluorescein diacetate (DCHF-DA) assay for quantifying the intracellular reactive oxygen species (ROSi) produced in four cultured cancer cell lines: drug-sensitive (K562) and drug-resistant (K562/adr) human erythromyelogenous leukemia cell lines, and drug-sensitive (GLC4) and drug-resistant (GLC4/adr) human small cell lung carcinoma cell lines. The oxidation of the probe to the fluorescent dichlorofluorescein (DCF) was continuously monitored by following the DCF fluorescence intensity as a function of time using a standard spectrofluorometer in the presence of an extracellular DCF fluorescence quencher (Co2+). By fitting the spectrofluorometric data to a kinetic model based on the following two reactions: (i) deacetylation of DCHF-DA to the oxidant-sensitive compound 2′,7′-dichlorofluorescein (DCHF) by cellular esterase enzymes (pseudo-first-order rate constant: ke) and (ii) oxidation of DCHF by ROSi (second-order rate constant: k2), the parameters intervening in DCF formation, ke and the product of k2 by the ROSi concentration, were quantitatively determined for the different cell lines studied. The results revealed that the intracellular esterase content or activity is similar in K562, K562/adr, and GLC4 cells, but 5-fold higher in GLC4/adr cells. The product k2[ROSi] was found to be similar in the four cell lines considered, with a mean value of (5.3±0.9)×10−7 cell−1 s−1. Assuming that H2O2 (in combination with peroxidases) is the primary responsible species for DCHF oxidation in intact cells, and using the rate constant value k 2 = 790 ± 62 M - 1 s - 1 established in our laboratory for the reaction of DCHF with H2O2 in the presence of horseradish peroxidase, the mean value of the intracellular levels of ROSi in those cells was estimated to be 0.67±0.16 nM per cell. Such a value compares favorably to H2O2 intracellular steady-state concentrations that have been estimated in the literature for a few other cell types.

Natural products from plants such as flavonoids are potential drugs to overcome multidrug resistance (MDR) in cancer treatments. However, their modes of action are still unclear. In this study, the effects of quercetin on mitochondrial membrane potential (ΔΨm) change as well as quercetin's ability to induce apoptosis and inhibit Pgp-mediated efflux of 99mTc-MIBI in K562/adr cells were investigated. Quercetin exhibits cytotoxicity against erythroleukemic cells: IC50 are 11.0 ± 2.0 µmol/L and 5.0 ± 0.4 µmol/L for K562 and K562/adr, respectively. Quercetin induces cell death via apoptosis in both K562 and K562/adr cells and does not inhibit Pgp-mediated efflux of 99mTc-MIBI. Quercetin (10 µmol/L, 3 h) and etoposide (100 µmol/L, 24 h) induce similar levels of apoptosis in K562 and K562/adr cells. Quercetin induces an increase followed by a decrease in |ΔΨm| value depending on its concentration. A decrease in the |ΔΨm| value is associated with an increase in the percentage of early apoptotic cells. It is clearly shown that quercetin results in a spontaneous ΔΨm change during apoptotic induction. Therefore, quercetin is potentially an apoptotic-inducing agent, which reacts at the mitochondrial level.Key words: multidrug resistance (MDR), quercetin, apoptosis, 99mTc-Annexin V, mitochondrial membrane potential (ΔΨm), 99mTc-MIBI.

We sought to determine the efficiency of the intracellular functional P-gp- and MRP1-mediated pumping of THP into acidic organelles in SiHa cells and etoposide-resistant SiHa/VP16 cells. The expression of both MDR1 and MRP1 genes of SiHa and SiHa/VP16 cells was clearly shown by using RT–PCR. The functional studies of both intracellular functional P-gp- and MRP1-mediated pumping were performed by using THP in a conventional spectrofluorometer, and they demonstrated that SiHa and SiHa/VP16 cells are good models to illustrate the functional role of intracellular P-gp and MRP1 in the transport of free cytosolic drug into acidic organelles. The functional P-gp and MRP1 proteins were identified both on plasma membranes and on intracellular vesicle membranes. Within the limit of experimental error, similar efficiencies in THP transport were observed in the two proteins at both locations in SiHa and SiHa/VP16 cells. The P-gp- and MRP1-mediated pump coefficient (kav), Michealis–Menten's constant (Kmv), and maximal pumping rate (Vmaxv) values of those located on vesicular membranes were 1.87 ± 0.30 pL·cell–1·s–1, 1.63 ± 0.21 μM, and 4.95 ± 0.45 nM·s–1, respectively. Drug retention inside acidic organelles (Cvmon) of SiHa cells was significantly higher than that of SiHa/VP16 cells, perhaps a consequence of slower movement of recycling endosomes and (or) lysosomes to the cell membrane of SiHa cells, leading to distended organelles and cell death. Our results suggest that intracellular P-gp and MRP1 proteins play an important role in the transport of free drug from cytosol to cytoplasmic acidic organelles.Key words: intrinsic resistance, multidrug resistance, intracellular functional P-glycoprotein and MRP1, acidic organelle, fluorescence spectroscopy, kinetic parameters.

Monte-Carlo simulations are used to investigate the effects of multiple ionization on the formation of HO 2 /O 2 − and H 2 O 2 in the radiolysis of water by 12 C 6+ ions up to ∼430 keV/μm, at neutral pH and 25 °C. Taking into account the double and triple ionizations of water molecules, the primary yields G HO 2 /O 2 − and G H 2 O 2 are calculated as a function of linear energy transfer (LET). Our results quantitatively reproduce the large increase observed in G HO 2 /O 2 − at high LET. They also simultaneously predict a slight maximum in G H 2 O 2 at ∼180–200 keV/μm, in excellent agreement with experiment. Under the conditions of this study, triple ionization is found to contribute only negligibly to G HO 2 /O 2 − .

In this work, we sought to investigate the relation existing between MDR1 mRNA levels, the resistance factor (RF), and the efficiency of efflux of pirarubicin (THP) mediated by P-glycoprotein (P-gp) in multidrug-resistant (MDR) K562 sublines. The MDR K562 sublines were selected from K562/adr cells by exposure to different adriamycin concentrations: 300 nM (K562/300), 1000 nM (K562/1000), and 10 000 nM (K562/10000), yielding RF values of 23.2, 26.5, and 39.6, respectively. The analysis of the P-gp encoding MDR1 gene overexpression by reverse transcriptase – polymerase chain reaction provided evidence of increased MDR1 mRNA levels when the adriamycin concentration used for the MDR cell selection increased. We used spectrofluorometric methods to determine the kinetics of the uptake and P-gp-mediated efflux of THP in the different selected MDR K562 sublines. Our data showed that (i) the maximal rate of P-gp-mediated efflux of THP, Vmax, increased with increasing RF; (ii) the observed Michaelis constant, Km, had the same value for all selected sublines, thus leading to an overall increase in the ratio Vmax/Km(5.1 × 10–3, 6.2 × 10–3, 6.8 × 10–3, and 9.3 × 10–3s–1for K562/adr, K562/300, K562/1000, and K562/10000 cells, respectively), and (iii) the determination of the Hill coefficient (nH) gave values close to 2, which suggested a positive cooperative transport of THP with the expelling of two molecules of THP per turnover of P-gp. This study demonstrated that, in the K562/adr sublines used in our experiments, P-gp played a major role in conferring the MDR phenotype. Moreover, under our experimental conditions, intracellular acidic organelles were shown to contribute to decreased drug–target interaction and, thereby, decreased cytotoxicity. The variation of the concentrations of THP accumulated in the acidic organelles as a function of the total THP concentration added to the cells was the same, within the limits of experimental errors, whatever the degree of resistance of the studied MDR K562 sublines. Finally, this study suggested that, in the selected MDR K562 sublines, the K+/H+antiporter exchanger could be activated by the pirarubicin transport, leading to a probable acidification of intracellular pH. The P-gp-mediated efflux of THP and an accumulation of THP in acidic organelles confer an advantage for MDR cells in surviving prolonged exposure to cytotoxic agents and giving rise to high degrees of resistance. Key words: multidrug resistance, P-glycoprotein, pirarubicin, acidic organelles, MDR1 mRNA levels, fluorescence spectroscopy, kinetic parameters.

A re-examination of our Monte-Carlo modeling of the radiolysis of liquid water by low linear-energy-transfer (LET ~ 0.3 keV µm–1) radiation is undertaken herein in an attempt to reconcile the results of our simulation code with recently revised experimental hydrated electron (e–aq) yield data at early times. The thermalization distance of subexcitation electrons, the recombination cross section of the electrons with their water parent cations prior to thermalization, and the branching ratios of the different competing mechanisms in the dissociative decay of vibrationally excited states of water molecules were taken as adjustable parameters in our simulations. Using a global-fit procedure, we have been unable to find a set of values for those parameters to simultaneously reproduce (i) the revised e–aq yield of 4.0 ± 0.2 molecules per 100 eV at "time zero" (that is, a reduction of ~20% over the hitherto accepted value of 4.8 molecules per 100 eV), (ii) the newly measured e–aq decay kinetic profile from 100 ps to 10 ns, and (iii) the time-dependent yields of the other radiolytic species H•, •OH, H2, and H2O2 (up to ~1 µs). The lowest possible limiting "time-zero" yield of e–aq that we could in fact obtain, while ensuring an acceptable agreement between all computed and experimental yields, was ~4.4 to 4.5 molecules per 100 eV. Under these conditions, the mean values of the electron thermalization distance and of the geminate electron–cation recombination probability, averaged over the subexcitation electron "entry spectrum," are found to be equal to ~139 Å and ~18%, respectively. These values are to be compared with those obtained in our previous simulations of liquid water radiolysis, namely ~88 Å and ~5.5%, respectively. Our average electron thermalization distance is also to be compared with the typical size (~64–80 Å) of the initial hydrated electron distributions estimated in current deterministic models of "spur" chemistry. Finally, our average probability for geminate electron–cation recombination agrees well with an estimated value of ~15% recently reported in the literature. In conclusion, this work shows that an adaptation of our calculations to a lower hydrated electron yield at early times is possible, but also suggests that the topic is not closed. Further measurements of the e–aq yields at very short times are needed. Key words: liquid water, radiolysis, electron–cation geminate recombination, electron thermalization distance, hydrated electron (e–aq), e–aq decay kinetics, time-dependent molecular and radical yields, Monte-Carlo simulations.

Monte-Carlo simulations are performed to calculate the temperature dependence of the primary hydrated electron yield (Geaq-) for liquid water irradiated by low linear-energy-transfer radiation (LET ~ 0.3 keV µm–1) in the range 25–325°C. Calculations are carried out by taking properly into account the effect of the time and temperature dependencies of the water dielectric constant on the electron–cation geminate recombination. Our computed Geaq- values slightly increase with increasing temperature, in good agreement with experiment. The product Geaq- εmax(eaq-), estimated by using existing experimental data of the maximum molar extinction coefficient εmax(eaq-), remains nearly constant or slightly increases, depending on the temperature dependence chosen for εmax. Our Geaq-εmax(eaq-) values compare generally well with most experimental data, as well as with the predictions of deterministic diffusion-kinetic model calculations. Moreover, our results indicate that the static dielectric constant of water (εs) does not play any significant role on the electron–cation recombination at early times. Such a finding is inconsistent with the interpretation, proposed by certain authors in the literature, that Geaq- should in fact decrease as temperature is increased because of an increased electron–cation geminate recombination due to a lowering of εs. Finally, the temperature dependence of the hydrated electron yields, calculated at various times between 10 ps and 1 µs, shows that at low LET, the time required to establish homogeneous chemistry in the bulk of the solution is ~10–6 s in the range ~25–100°C, and that this time diminishes to ~10–7 s at higher temperatures. Key words: liquid water, radiolysis, temperature, hydrated electron (eaq-), radiolytic yields, electron–cation geminate recombination, dielectric constant, molar extinction coefficient of eaq-, homogenization time.

Monte-Carlo simulations are used to calculate the primary yield of hydrogen peroxide (GH2O2) of the radiolysis of pure, deaerated liquid water as a function of linear energy transfer (LET) of the incident radiation over the range ~0.3–100 keV µm–1, at 25 and 300°C. The radiations include 1H+, 2H+, 4He2+, 7Li3+, and 12C6+ ions with energies from 0.17 MeV to 3.6 GeV. At 25°C, it is found that our GH2O2 values, calculated with protons of different initial energies, show a monotonic increase as a function of LET, in agreement with the commonly assumed expectation of an increase in molecular yields with increasing LET. Our calculated H2O2 yields at 300°C increase significantly faster with LET than do their corresponding 25°C values, showing that the temperature dependence of GH2O2 at higher LET is less than for low-LET radiation. We also report our results on the temporal variations of the H2O2 yields, in the interval ~1 × 10–13 – 1 × 10–6 s, at 25 and 300°C and for the different types of radiation considered. Finally, we find that for incident ions of equal LET > 10 keV µm–1, GH2O2 decreases as the ion velocity increases, from protons (or deuterons) to carbon ions. These differences produced in GH2O2 by changing the type of radiation are explained by the greater mean energy of secondary electrons from the higher velocity ions, which penetrate to a greater average distance from the actual particle track, with a corresponding decrease in molecular yields. Our calculated GH2O2 values compare generally well with the experimental data available from the literature and are also in good accord with the predictions of deterministic diffusion-kinetic model calculations reported earlier.Key words: liquid water, radiolysis, primary yields, hydrogen peroxide (H2O2), linear energy transfer (LET), accelerated protons and heavy ions, temperature, Monte-Carlo simulations.

Overcoming MDR (multidrug resistance) phenomena is a crucial aspect of cancer chemotherapy research. Artemisinin and its derivatives have been found to inhibit the proliferation of cancer cells in the microM range. They poorly inhibited the function of P-glycoprotein and did not inhibit the function of MRP1-protein. The concentrations required to inhibit by 50% the function of P-glycoprotein are 110+/-5 microM. Artemisinin, artesunate and dihydroartemisinin efficiently decreased the mitochondrial membrane potential, leading to a decrease in intracellular ATP in all cell lines tested: by 30 to 50% at 5 microM. Artemisinin, artesunate and dihydroartemisinin increased cytotoxicity of pirarubicin and doxorubicin in P-glycoprotein-overexpressing K562/adr, and in MRP1-overexpressing GLC4/adr, with the delta(0.5) ranging from 200 to 860 nM, but not in their corresponding drug-sensitive cell lines. In this range of concentrations these compounds did not decrease the function of P-glycoprotein, suggesting a mechanism by which the drugs did not reverse MDR phenomenon at the P-glycoprotein level but at the mitochondrial level.

Resistance to chemotherapeutic agent is a major cause of treatment failure in patients with cancer. In many cases, the primaly mechanism leading to a multidrug-resistant phenotype is the plasma-membrane localized overexpression of drug efflux transporters, such as P-glycoprotein. However, acidic intracellular organelles seem also to participate in resistance to chemotherapeutic drugs and the determination of the pH of these organelles is of importance. In the present study we have used a new fluorescent derivative of verapamil, 2-2-dipheny1-5-[(methylaminomethyl)anthracene] pentanenitrile (EDP 96), and show that it is an efficient inhibitor of the P-gp-mediated efflux of anthracycline in K562 resistant cells. The fluorescence of EDP 96 is environmental and pH sensitive. EDP 96 is a weak base (pKa=6.0) and its aceumulation into K562 cells is accompanied by a significant fluorescence increase due to its entry of the drug into acidic regions in the cells. We have used this properties to develop a new method to accurately determine the pH of acidic organelle.

Monte-Carlo simulations are used to calculate the primary radical and molecular yields (g-values) of water radiolysis versus temperature in the range 25–300°C, for different high linear energy transfer radiations (protons, 2 H + , and 7 Li 3+ ) up to ∼ 65 keV / μm . The results are compared, at ∼10−7 s, to experimental data at 25°C, 95°C, and 180°C. Excellent agreement is obtained for g(e−aq) and g( OH ) over the whole temperature range, but g(H2), [g(H )+g(H2)], and g(H2O2) are substantially larger than the measured values at 180°C. Our g-values are also compared with fast-neutron radiolysis yields and with deterministic modeling calculations. The agreement is generally satisfactory.

Monte-Carlo simulations are used to calculate the primary yield of hydrogen atoms (GH ) of water radiolysis versus linear energy transfer (LET) up to ∼100 keV/μm, at 25°C and 300°C. The GH values calculated at 25°C using protons show a maximum near 6.5 keV/μm before decreasing steeply at higher LET. At 300°C, GH remains constant below ∼7 keV/μm, while at higher LET it decreases faster than do its corresponding 25°C values. For different radiation types (protons, 2 H + , 4 He 2+ , 7 Li 3+ , and 12 C 6+ ) of equal LET>10 keV/μm, GH increases as the incident ion velocity increases. The results compare generally well with experiment.

Meesungnoen, J., Benrahmoune, M., Filali-Mouhim, A., Mankhetkorn, S. and Jay-Gerin, J-P. Monte Carlo Calculation of the Primary Radical and Molecular Yields of Liquid Water Radiolysis in the Linear Energy Transfer Range 0.3–6.5 keV/μm: Application to 137Cs Gamma Rays. Monte Carlo simulations of the radiolysis of neutral liquid water and 0.4 M H2SO4 aqueous solutions at ambient temperature are used to calculate the variations of the primary radical and molecular yields (at 10–6 s) as a function of linear energy transfer (LET) in the range ∼0.3 to 6.5 keV/μm. The early energy deposition is approximated by considering short (∼20–100 μm) high-energy (∼300–6.6 MeV) proton track segments, over which the LET remains essentially constant. The subsequent nonhomogeneous chemical evolution of the reactive species formed in these tracks is simulated by using the independent reaction times approximation, which has previously been used successfully to model the radiolysis of water under various conditions. The...

To our knowledge, this is the first study to investigate the modification of P-glycoprotein functionality in living resistant cells after photolabeling. For this purpose, four new photoactive verapamil analogues were synthesized. These compounds have the same efficacy as verapamil to increase pirarubicin (pira) incorporation into living multidrug resistant (MDR) K562 cells and to sensitize them to the cytotoxic effect of this anthracycline derivative, indicating that they act as typical MDR modifiers in MDR cells. These compounds were used to photolabel P-glycoprotein (P-gp) in living resistant cells. Irradiation did not result in photodamage to cells, and P-gp functionality was verified by the ability of living cells to incorporate pira. The irradiation of resistant cells, 106/mL, in the presence of a verapamil analogue at concentrations equal to or higher than 3 μM yielded 70% inhibition of P-gp functionality. Our data provide the first evidence that the binding of a verapamil analogue to P-gp is not sufficient to completely inhibit the efflux of this anthracycline. The cells were, subsequently, cultured for several days. Resistance was progressively recovered with time, with the treated cells being just as resistant as before photolabeling after 6 days.

The new potential tracer of bone imaging, AHBDP-Sn(II)-TcO.3H2O was synthesized by reducing the TcO4− to TcO2+ in the presence of AHBDP and Sn(ll)’s reducing agent. We found that tin rapidly forms a stable complex with AHBDP, giving AHBDP-Sn(II).3H2O. In the excess of AHBDP-Sn(ll).3H2O, the AHBDP-Sn(II).3H2O coordinates with TcO2+ to give AHBDP-Sn(II)-TcO.3H2O which could polymerise or oligomerise to give hydrophobic species. The overall process appears as a first-order reaction (K= 0.67 ± 0.005s−1). In rats, the fixation of AHBDP-Sn(II)-99m TcO. 3H2O on bone is homogeneous and the scintigraphic images have the same quality as those of 1-hydroxymethane-1,1-diphosphonate-Technetium (HMDP-99mTc). The activity in non-target organs was neglible.

The number of patients with cancer is increasing. New therapeutic agents to overcome drug-resistant tumors are urgently needed. Cyrtosperma johnstonii N.E. Br. (Araceae) is used for treatment of cancer in Thai traditional medicine. This study aimed to evaluate antioxidant activity and cytotoxicity of C. johnstonii extracts on human cancer cells.Dried powder of C. johnstonii rhizomes was extracted with several solvents. The 0.1 mg/ml extract solution was tested for antioxidant activity by 2,2'-azinobis-3-ethylbenzothiazoline-6-sulfonic acid (ABTS) and ferric reducing antioxidant power (FRAP) assays. Color formation from 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide was used to determine cell viability. Standardization of the extract was performed by high-performance liquid chromatography (HPLC) with photodiode array detector at 254 and 360 nm. Cell cycle arrest was evaluated by flow cytometry after 5 min, 12 h and 24 h treated with 20 µg/ml of the acetone extract.The acetone extract exhibited the highest phenolic content and antioxidant activity (TEAC and EC values = 19.2 ± 0.14 and 19.2 ± 0.31 mM/mg, respectively). The IC₅₀ values for leukemia ranged from 11 ± 1 to 29 ± 3 µg/ml and from 5 ± 2 to 6 ± 0 µg/ml for small cell lung carcinoma cells. Cell cycle arrest occurred at the G2/M phase followed by apoptosis. HPLC analysis revealed that rutin is the major constituents of the extract.The acetone extract of C. johnstoni is a promising source of natural antioxidants and anticancer. The extract inhibits cancer cells effectively with less effect on normal cells.

The oxidation of sodium diethyldithiocarbamate (DDC) by hydrogen peroxide or superoxide radicals has been investigated. Hydrogen peroxide oxidizes DDC, leading to the formation of a hydrated form of disulfiram, a dimer of DDC having a disulfide group. In equimolar conditions, the overall process appears as a first-order reaction (k = 0.025±0.005 s −1 ), the first step being a second-order reaction (k = 5.0±0.1mol −1 .1. s −1 ). No radical intermediate was observed in this process. In the presence of an excess of any of the reagents, the hydrated form of disulfiram transforms into different products corresponding to the fixation of oxygen by sulfur atoms or replacement of C = S group by ketone function, in the presence of an excess of hydrogen peroxide. Superoxide anions (produced by steady-state 60 Co γ-radiolysis) oxidize DDC, yielding similar products to those obtained with hydrogen peroxide with a maximum oxidation G-value of 0.3 μmol.J −1 . The rate constant k(O 2 ·− + DDC) is equal to 900 mol −1 . 1. s −1 .

Xanthone exhibits several medicinal activities and especially it inhibits the growth of cancer cells. However, the use of xanthone is limited because of its low aqueous solubility and systemic toxicity. In the present study xanthone was loaded into poly(ethylene glycol)-b-poly[N-(2-hydroxypropyl) methacrylamide-dilactate] mPEG-b-p(HPMAm-Lac(2)) micelles in order to overcome these drawbacks. It was shown that xanthone could be loaded in these micelles up to 2 mg/mL with ~100% entrapment efficiency and ~20% loading capacity. The average particle diameter of the xanthone loaded mPEG-b-p(HPMAm-Lac(2)) micelles as determined by dynamic light scattering ranged from 84 to 112 nm. In vitro assays showed that xanthone in its free form as well as loaded in polymeric micelles had a high cytotoxicity towards both doxorubicin sensitive and, importantly, resistant cancer cells. On the other hand empty mPEG-b-p(HPMAm-Lac(2)) micelles did not show any cytotoxicity towards normal cells (PBMCs). Interestingly, the cytostatic effect of xanthone towards normal cells was masked when loaded in the micelles. The mechanism of cell growth inhibition by xanthone-loaded polymeric micelles was mediated through induction of apoptosis, as evidenced from a subdiploid peak of propidium iodide stained cells using flow cytometric analysis. From the results of this study it can be concluded that xanthone has potent anticancer activity not only on sensitive but also on doxorubicin resistant cancer cell lines. mPEG-b-p(HPMAm-Lac(2)) micelles are therefore attractive delivery systems of xanthone for the treatment of cancer.

Background Multidrug resistance (MDR) is a major obstacle in cancer treatment and is often the result of overexpression of the drug efflux protein, P-glycoprotein (P-gp), as a consequence of hyperactivation of NFκB, AP1 and Nrf2 transcription factors. In addition to effluxing chemotherapeutic drugs, P-gp also plays a specific role in blocking caspase-dependent apoptotic pathways. One feature that cytotoxic treatments of cancer have in common is activation of the transcription factor NFκB, which regulates inflammation, cell survival and P-gp expression and suppresses the apoptotic potential of chemotherapeutic agents. As such, NFκB inhibitors may promote apoptosis in cancer cells and could be used to overcome resistance to chemotherapeutic agents. Results Although the natural withanolide withaferin A and polyphenol quercetin, show comparable inhibition of NFκB target genes (involved in inflammation, angiogenesis, cell cycle, metastasis, anti-apoptosis and multidrug resistance) in doxorubicin-sensitive K562 and -resistant K562/Adr cells, only withaferin A can overcome attenuated caspase activation and apoptosis in K562/Adr cells, whereas quercetin-dependent caspase activation and apoptosis is delayed only. Interestingly, although withaferin A and quercetin treatments both decrease intracellular protein levels of Bcl2, Bim and P-Bad, only withaferin A decreases protein levels of cytoskeletal tubulin, concomitantly with potent PARP cleavage, caspase 3 activation and apoptosis, at least in part via a direct thiol oxidation mechanism. Conclusions This demonstrates that different classes of natural NFκB inhibitors can show different chemosensitizing effects in P-gp overexpressing cancer cells with impaired caspase activation and attenuated apoptosis.

We sought to investigate the early biological effects of chlorpyrifos among 53 Thai fruit farm workers by measuring the plasma cholinesterase (PChE) and red blood cell cholinesterase (AChE) activities, a biomarker of organophosphate (OPs) pesticide during one fruit crop. The ChE activity (V(m)/K(m)) was spectrophotometrically analyzed before and after exposing to chlorpyrifos. The V(m)/K(m) values of both non-spraying and spraying seasons are found as normal distribution pattern. The median PChE and AChE activities among farm workers in the non-spraying season were 2.3 x 10(-3) s(-1) and 7.26 x 10(-5) s(-1), respectively. The median PChE and AChE activities of the farm workers in the spraying season were 2.02 x 10(-3) s(-1) and 5.95 x 10(-5) s(-1), respectively. The mean V(m)/K(m) values of PChE shifted left (t-test, p=0.013), indicating a decrease in PChE activity in the farm workers exposed to chlorpyrifos. However, the V(m)/K(m) values of AChE in nonspraying season and in the spraying season were not different (t-test, p=0.246). We propose that PChE activity can be used as a biomarker for monitoring early toxicity induced by chlorpyrifos insecticide.

5-Aryl-3-(4-hydroxyphenyl)-1,3,4-oxadiazole-2(3H)-thiones 3 were prepared by cyclocondensation of 1-(4-hydroxyphenyl)-2-aroylhydrazines with thiophosgene. All compounds exhibited antiproliferation activity in K562, IC(50) ranging from 24 to 94 micro M comparable efficacy with apigenin and genistein and showed more potent antiproliferation of K562/adr cells, highly expressing P-glycoprotein. Compounds 3g, 3e and 3a inhibited the function of P-glycoprotein with the alpha(0.5) equal to 10+/-3 micro M, 21+/-5 micro M and 34+/-7 micro M, respectively.

Meesungnoen, J., Jay-Gerin, J-P., Filali-Mouhim, A. and Mankhetkorn, S. Low-Energy Electron Penetration Range in Liquid Water. Radiat. Res. 158, 657–660 (2002). Monte Carlo simulations of electron tracks in liquid water are performed to calculate the energy dependence of the electron penetration range at initial electron energies between 0.2 eV and 150 keV, including the subexcitation electron region (

Artemisinin, a sesquiterpene with endoperoxide bond, possesses potent antimalarial activity against the ring and late stage of chloroqine-resistant Plasmodium falciparum malaria both in vitro and in vivo. The mode of antimalarial activity of artemisinin is iron-dependent. The aim of this study was to investigate the reactions of artemisinin with ferrous and ferric ions in aqueous buffer. Artemisinin generated a cycle of iron oxidation and reduction. It oxidized ferrous and reduced ferric ions with similar rate of reaction (k=10+/-0.5 M(-1) x s(-1) for ferrous and k=8.5+/-2.0 M(-1) x s(-1) for ferric ion). The major active product was dihydroartemisinin which exhibited antimalarial activity at least 3 times more potent than artemisinin. Dihydroartemisinin preferably binds to ferric ion, forming ferric-dihydroartemisinin complex. The re-oxidation of the complex gives artemisinin and ferric ion. This suggests that in aqueous buffer, the reaction of artemisinin with iron may give rise to the active reaction products, one of them being dihydroartemisinin, which is responsible for antimalarial activity.

P-glycoprotein (P-gp) is a membranous ATPase responsible for the multidrug resistance phenotype. The effect on P-gp-mediated transport of anthracyclines of cell irradiation in the presence of 2,2-diphenyl-5-[N-1-(o-azidophenyl)ethylamino]valeronitrile (VP*), a photoactivable analogue of verapamil was studied in viable K562/ADR cells. The derivatives were daunorubicin (DNR), idarubicin (IDA), 8-(S)-fluoro-idarubicin (F-IDA), 2′-bromo-4′-epi-daunorubicin (Br-DNR) and pirarubicin (PIRA). It was observed that the irradiation in the presence of the verapamil analogue was unable to completely inhibit the P-gp-mediated efflux of anthracyclines and we estimated that P-gp retained 10–20% of its ability to pump these toxins. The ability of verapamil, DNR, IDA, F-IDA, Br-DNR and PIRA to inhibit the effect of VP* was studied. For this purpose, cells were irradiated in the presence of VP* and various concentrations of either verapamil or of one of the anthracyclines and then the P-gp functionality was checked by its ability to pump pirarubicin. It was observed that (i) the effect observed, when cells were irradiated in the presence of VP*, was completely blocked by the presence of verapamil; (ii) that anthracyclines are able to partially inhibit the VP* effect. This inhibition occurs at low concentration of anthracycline and depends on the nature of the derivative used. With those used in that study, after the photoirradiation of K562/ADR cells in the presence of VP* and anthracycline, P-gp has retained 50±5% of its functionality. The anthracycline concentration required for this inhibition is rather low, the total drug concentration yielding 50% of the effect ranged from 0.5 (Br-DNR) to 4 μM (F-IDA). The corresponding cytosolic concentrations are highly correlated with the values of Km determined previously.

The basic distinguishing feature of all cells expressing functional P-glycoprotein-multidrug resistance is a decrease of steady state drug levels as compared to those in drug-sensitive controls. A variety of small molecules, such as verapamil and cyclosporin A, bind to P-glycoprotein and inhibit its ability to pump out antitumor drugs. The kinetics of P-glycoprotein-mediated efflux of various anthracycline derivatives was measured in multidrug-resistant (MDR) K562 cells in the presence of verapamil. Used for the purpose were daunorubicin, idarubicin and 8-S-fluoro-idarubicin which have the same pKa of deprotonation equal to 8.4, but different lipophilicity, 4'-epi-2'-bromo-daunorubicin which has a lipophilicity which is comparable to that of daunorubicin but a pKa equal to 6.3, pirarubicin with pKa equal to 7.7 and lipophilicity different from that of these derivatives were used. Our data show (1) that verapamil is unable to completely block the P-glycoprotein-mediated efflux of anthracyclines and that 10% of its functionality remains even with high verapamil concentrations, (2) that the ability of verapamil to restore intracellular accumulation of anthracyclines in MDR cells depends on the kinetics of their uptake. With fast kinetics uptake, as is the case for idarubicin, 8-S-fluoro-idarubicin, 4'-epi-2'-bromo-daunorubicin and pirarubicin (which have either a low pKa and/or high lipophilicity), verapamil can restore in multidrug resistant cells an intracellular drug level which is comparable to that observed in sensitive cells. This is not possible when the kinetics of uptake is low as is the case for daunorubicin. Cyclosporin A is a more potent modulator and is able to fully restore daunorubicin accumulation in multidrug resistant cells.

We sought to investigate the apoptosis-inducing activities of quercetin, Siamois 1, and Siamois 2 against invasive estrogen-receptor negative MDA-MB 435 cells xenografted in athymic nude mice. This study clearly demonstrated that these compounds exhibited apoptosis-inducing activities in cell culture system. Quercetin (20 microg/mL), Siamois 1 (100 microg/mL), and Siamois 2 (200 microg/mL) can induce apoptotic cell death by 40 +/-5%, 44 +/- 14 %, and 31 +/- 13 %, respectively. Two-fold of IC50 of these compounds were clearly found to induce apoptosis in breast tumor tissue which can be determined by 99mTc-Annexin V scintigraphy and histological staining. This is the first report that the apoptosis-inducing effects of quercetin, Siamois 1 and Siamois 2 on the MDA-MB 435 cell in vitro were effectively extrapolated to the in vivo situation. These compounds might be considered as a simple dietary supplement and with further clinical investigation for their use as a nutrition-based intervention in breast cancer treatment.

Problem statement: Quercetin and its glycoside derivatives are increasingly receiving interests as new generation of anticancer molecules and were recognized by multidrug resistant transporters such as P-glycoprotein and MRP1 protein. Of relevance to their use as anticancer agents alone or in combination with other agents, this study aims to analyze the interaction of the compounds with the MDR transporters including P-glycoprotein and MRP1 protein in living multidrug resistant cells. Approach: The potential MDR reversing action of flavonoids was assessed by using the co-treatment of anticancer drug, pirarubicin or daunorubicin and quercetin, quercetrin or rutin compared with the series of co-treatment of pirarubicin or daunorubicin and the known inhibitor such as cyclosporine A and verapamil. The evidence of direct interaction of molecules with MDR protein was investigated by measuring the ability of inhibition of the rate of P-glycoprotein- and MRP1-mediated efflux of pirarubicin out of cells. Results: Quercetin and its glycoside derivatives efficiently inhibited cancer cell proliferation and re-sensitize the MDR cells to pirarubicin but not for daunorubicin. Our results clearly show that quercetin, quercetrin except rutin non-competitively inhibited the function of P-glycoprotein in K562/adr and MRP1 in GLC4/adr cells. The determined KI value of P-glycoprotein was equal to 0.33 µM for quercetin and 1 µM for quercetrin and KI value of MRP1 was equal to 0.45 mM for quercetin and 0.5 mM for quercetrin. Conclusion: The overall results demonstrated that quercetin, quercetrin and rutin should be considered as potential pharmaceutical molecules that might be used as MDR inhibitors.