Antioxidant activity and cytotoxicity of Cyrtosperma johnstonii extracts on drug sensitive and resistant leukemia and small cell lung carcinoma cells.

CONTEXT: 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. MATERIALS AND METHODS: 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. RESULTS: 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. DISCUSSION AND CONCLUSION: 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.

Evidence of Nrf2/Keap1 Signaling Regulation by Mitochodria-Generated Reactive Oxygen Species in RGK1 Cells.

It has been known that reactive oxygen species (ROS) are generated from the mitochondrial electron transport chain (ETC). Majima et al. proved that mitochondrial ROS (mtROS) caused apoptosis for the first time in 1998 (Majima et al. J Biol Chem, 1998). It is speculated that mtROS can move out of the mitochondria and initiate cellular signals in the nucleus. This paper aims to prove this phenomenon by assessing the change in the amount of manganese superoxide dismutase (MnSOD) by MnSOD transfection. Two cell lines of the same genetic background, of which generation of mtROS are different, i.e., the mtROS are more produced in RGK1, than in that of RGM1, were compared to analyze the cellular signals. The results of immunocytochemistry staining showed increase of Nrf2, Keap1, HO-1 and 2, MnSOD, GCL, GST, NQO1, GATA1, GATA3, GATA4, and GATA5 in RGK1 compared to those in RGM1. Transfection of human MnSOD in RGK1 cells showed a decrease of those signal proteins, suggesting mtROS play a role in cellular signals in nucleus.

Differential chemosensitization of P-glycoprotein overexpressing K562/Adr cells by withaferin A and Siamois polyphenols.

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 NFkappaB, 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 NFkappaB, which regulates inflammation, cell survival and P-gp expression and suppresses the apoptotic potential of chemotherapeutic agents. As such, NFkappaB 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 NFkappaB 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 NFkappaB inhibitors can show different chemosensitizing effects in P-gp overexpressing cancer cells with impaired caspase activation and attenuated apoptosis.

Delivery of Superparamagnetic Polymeric Micelles Loaded With Quercetin to Hepatocellular Carcinoma Cells.

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.

Quercetin, Siamois 1 and Siamois 2 induce apoptosis in human breast cancer MDA-mB-435 cells xenograft in vivo.

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.

3D modeling of keloid scars in vitro by cell and tissue engineering.

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.

Visualization of Inflammation at Early Stage of Lung Cancer in Xenografted Temporally Immunosuppression Rats by Ferrioxamine Magnetic Resonance Imaging.

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-1 of 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 the in vivo imaging, 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.

Low-energy electron penetration range in liquid water.

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 (<7.3 eV). Our calculated electron penetration distances are compared with available experimental data and earlier calculations as well as with the results of simulations using newly reported amorphous ice electron scattering cross sections in the range approximately 1-100 eV.

Rhodamine B as a mitochondrial probe for measurement and monitoring of mitochondrial membrane potential in drug-sensitive and -resistant cells.

In order to get more insight into the energetic state of multidrug-resistance (MDR) cell compared with its corresponding sensitive cell, a noninvasive fluorescence method for determining and monitoring the mitochondrial membrane potential (DeltaPsi(m)), using rhodamine B and 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide (MTT) was established. Rhodamine B distributes across biological membranes in response to the electrical transmembrane potential. P-glycoprotein- and MRP1-protein-mediated efflux do not create a concentration gradient, leading the cell-rhodamine B system to reach a steady state, where the ratio of cytosolic to extracellular rhodamine B was equal to 1. The mitochondrial matrix rhodamine B concentration was precisely determined as a decrease of rhodamine B fluorescence in the presence of formazan, a rhodamine B fluorescence quencher, which locally accumulates in the matrix of mitochondria. The kinetics of decrease in rhodamine B fluorescence (V(i)) can be used to estimate DeltaPsi(m) using the Nernst equation: DeltaPsi(m)=-61.54 log V(i)-258.46. The DeltaPsi(m) values determined were -160+/-4 mV for K562 cell, -146+/-6 mV for K562/adr cell, -161+/-10 mV for GLC4 cell and -168+/-2 mV for GLC4/adr cell. An increase or a decrease in DeltaPsi(m) consequently followed an increase or a decrease in the cellular ATP contents. An increase ATP content in the two MDR cell lines can protect cells from cytotoxicity induced by pirarubicin.

Cytostatic effect of xanthone-loaded mPEG-b-p(HPMAm-Lac2) micelles towards doxorubicin sensitive and resistant cancer cells.

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.

PEG-OCL micelles for quercetin solubilization and inhibition of cancer cell growth.

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.

Spectrophotometric determination of plasma and red blood cell cholinesterase activity of 53 fruit farm workers pre- and post-exposed chlorpyrifos for one fruit crop.

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.

Modulation of multidrug resistance by artemisinin, artesunate and dihydroartemisinin in K562/adr and GLC4/adr resistant cell lines.

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.

Functional study of intracellular P-gp- and MRP1-mediated pumping of free cytosolic pirarubicin into acidic organelles in intrinsic resistant SiHa cells.

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 (k v a), Michealis-Menten's constant (K V m), and maximal pumping rate (V V max) values of those located on vesicular membranes were 1.87 +/- 0.30 pL x cell-1 x s-1, 1.63 +/- 0.21 microM, and 4.95 +/- 0.45 nM x s-1, respectively. Drug retention inside acidic organelles (C mon V) 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.

Synthesis and evaluation of 5-Aryl-3-(4-hydroxyphenyl)-1,3,4-oxadiazole-2-(3H)-thiones as P-glycoprotein inhibitors.

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.

Relation between MDR1 mRNA levels, resistance factor, and the efficiency of P-glycoprotein-mediated efflux of pirarubicin in multidrug-resistant K562 sublines.

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), 1,000 nM (K562/1,000), and 10,000 nM (K562/10,000), 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 x 10(-3), 6.2 x 10(-3), 6.8 x 10(-3), and 9.3 x 10(-3) s(-1) for K562/adr, K562/300, K562/1,000, and K562/10,000 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 TFP 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.