Comparative toxicity of fostriecin,hepsulfam and pyrazine diazohydroxide to human and murine hematopoietic progenitor cells in vitro

Summary The in vitro myelotoxic potentials of three investigational antitumor agents, Fostriecin, Hepsulfam and pyrazine diazohydroxide (PZDH), were evaluated utilizing clonogenic assays. Human and murine marrow cells were exposed to each drug for 1 hr prior to culture in microcapillary (human) or Petri dish (murine) assays. Fostriecin (0.22–220 M), Hepsulfam (0.34–340 M) and PZDH (0.68–680 M) inhibited myeloid (CFU-gm), erythroid (BFU-e, CFU-e) and megakaryocytic (CFU-meg) colony formation in a concentration-dependent manner. CFU-e from both species were more sensitive to Fostriecin than the other progenitors and murine cells more sensitive overall to Fostriecin than their human counterparts. Murine CFU-e were also more sensitive to Hepsulfam than human CFU-e, with CFU-gm and BFU-e being similarly affected in both species. Human BFU-e were greatly inhibited by PZDH, whereas murine BFU-e were relatively resistant to its toxic effects. Fostriecin was the most toxic of the three antitumor agents, with PZDH the least toxic.     

AG2034: a novel inhibitor of glycinamide ribonucleotide formyltransferase

Summary The glycinamide ribonucleotide formyltransferase (GARFT) inhibitor, 4-[2-(2-amino-4-oxo-4,6,7,8-tetrahydro-3H-pyrimidino[5,4-6][1,4]thiazin-6-yl)-(S)-ethyl]-2,5-thienoyl-L-glutamic acid (AG2034), was designed from the X-ray structure of the GARFT domain of the human tri functional enzyme. AG2034 inhibits human GARFT (K_i = 28 nM), has a high affinity for the folate receptor (K _d = 0.0042 nM), and is a substrate for rat liver folylpolyglutamate synthetase (K _m = 6.4 μM, V _max = 0.48 nmole/hr/mg). The IC₅₀ for growth inhibition was 4 nM against L1210 cells and 2.9 nM for CCRF-CEM cells in culture. In vitro growth inhibition can be reversed by addition of either hypoxanthine or AICA (5-aminoimidazole-4-carboxamide) to the culture medium. A cell line with impaired transport of reduced folates, L1210/CI920 [1], was resistant to AG2034 indicating that this compound can enter cells by utilizing the reduced folate carrier. AG2034 showed in vivo antitumor activity against the 6C3HED, C3HBA, and B-16 murine tumors and in the HxGC₃, KM20L2, LX-1, and H460 human xenograft models, and has been selected for preclinical development towards clinical trials.     

Cyclopropyl Carboxamides: A New Oral Antimalarial Series Derived from the Tres Cantos Anti-Malarial Set (TCAMS)

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Human CD34+ progenitor hematopoiesis in liquid culture for in vitro assessment of drug-induced myelotoxicity

Utilization of validated CFU-GM assays for myelotoxicity screening is hampered by its labor-intensive and low-throughput nature. Herein, we transformed the defined CFU-GM assay conditions and IC₉₀ endpoint into a higher throughput format. Human CD34⁺ hematopoietic progenitors were cultured in a 96-well plate for 14 days with the same cytokine (rhGM-CSF) used in the CFU-GM assay. Expansion and differentiation toward myeloid lineages were manifested by characteristic changes in nuclear and cytoplasmic morphology and by temporal expression patterns of CD34, CD11b and CD13 markers. Inhibition of CD34⁺ cell myelopoiesis by 12 anticancer drugs known to induce myelotoxicity in the clinic was quantifiable using either general cytotoxicity endpoints (cell growth area or total nucleus count) or lineage specific readouts (count of cells expressing CD11b and/or CD13). The IC₅₀ and IC₉₀ values derived from the concentration-response curves of 14-day drug exposure in CD34⁺ cell culture were highly correlated with those from the international validation study of the CFU-GM assay, demonstrating capability to assess general cytotoxicity, cell proliferation and myelopoiesis simultaneously. These results suggest that this human CD34⁺ hematopoietic progenitor cell assay can be used as a direct replacement for the validated, low throughput CFU-GM assay, and could expand application of in vitro myelotoxicity testing.     

FCE 24517-resistant MCF-7 human breast cancer cell line: selection and characterization.

We have developed a stable line of the human breast carcinoma cell line MCF-7 by in vitro continuous exposure to increasing concentrations of the antitumoral alkylating agent FCE 24517 (tallimustine). The selected line, MCF-7/24517(1), was resistant to the selecting agent (RI=10) and to a lesser degree to melphalan, MEN 10710 (a related dystamycin analog), doxorubicin and etoposide, but not to m-AMSA. MCF-7/24517(1) cells did not express the multidrug-resistant phenotype, evaluated in terms of mRNA for mdr-1 and gp170 glycoprotein. A significant, albeit modest, increase in the cellular content of glutathione was measured and therefore other resistance mechanism(s) should be operative. We conclude that the MCF-7/24517(1) line is a valuable model to investigate the mechanisms of resistance of FCE 24517 and its derivatives.     

Comparative in vitro and ex-vivo myelotoxicity of aflatoxins B1 and M1 on haematopoietic progenitors (BFU-E,CFU-E,and CFU-GM): Species-related susceptibility

Haemato- and myelotoxicity are adverse effects caused by mycotoxins. Due to the relevance of aflatoxins to human health, the present study, employing CFU-GM-, BFU-E- and CFU-E-clonogenic assays, aimed at (i) comparing, in vitro, the sensitivity of human vs. murine haematopoietic progenitors to AFB1 and AFM1 (0.001–50 μg/ml), (ii) assessing whether a single AFB1 in vivo treatment (0.3–3 mg/kg b.w.) alters the ability of murine bone marrow cells to form myeloid and erythroid colonies, and (iii) comparing the in vitro with the in vitro ex-vivo data.We demonstrated (i) species-related sensitivity to AFB1, showing higher susceptibility of human myeloid and erythroid progenitors (IC₅₀ values: about 4 times lower in human than in murine cells), (ii) higher sensitivity of CFU-GM and BFU-E colonies, both more markedly affected, particularly by AFB1 (IC₅₀: 2.45 ± 1.08 and 1.82 ± 0.8 μM for humans, and 11.08 ± 2.92 and 1.81 ± 0.20 μM for mice, respectively), than the mature CFU-E (AFB1 IC₅₀: 12.58 ± 5.4 and 40.27 ± 6.05 μM), irrespectively of animal species, (iii) regarding AFM1, a species- and lineage-related susceptibility similar to that observed for AFB1 and (iv) lack of effects after AFB1 in vivo treatment on the proliferation of haematopoietic colonies.     

AN IN VITRO STUDY OF INHIBITORY ACTIVITY OF GOSSYPOL, A COTTONSEED EXTRACT, IN HUMAN CARCINOMA CELL LINES

Gossypol, a cottonseed extract, has been shown to have antiproliferative activity in a variety of cancer cell lines. The objective of this study was to determine the inhibitory effects of gossypol on cell proliferation. Five human carcinoma cell lines were evaluated including endometrial (RL95-2), ovarian (SKOV-3), medullary thyroid (TT), and adrenocortical (NCI-H295R and SW-13). Gossypol and the metabolite, apogossypol hexaacetate, were examined at concentrations up to 500 μg ml⁻¹ and the IC₅₀ was determined using the MTT assay. Gossypol and apogossypol hexaacetate produced a dose-dependent growth inhibition in all cellular lines examined. The IC₅₀ for gossypol ranged from 1.3 to 18.9 μM while the IC₅₀ for apogossypol hexaacetate ranged from 5.2 to 9.0 μM. The results indicate that gossypol possesses antiproliferative action toward human carcinoma cells in vitro. These investigations suggest that gossypol may have therapeutic potential for the treatment of cancer.     

Characterization of the toxicity of distamycin derivatives on cancer cell lines and rat heart.

The cytotoxicity and cardiotoxicity of benzoyl mustard (FCE 24517) and epoxamido (FCE 24561) synthetic derivatives of distamycin A were reported in the present study. The 50% inhibiting concentration (IC50) of colony formation of FCE 24517 on human SNB-19 glioblastoma, A2780 ovarian cancer and DU 145 prostate cancer was at least three times lower than that of FCE 24561; on the same cell lines the IC50 of DXR was up to 14 and 240 times higher than that of FCE 24561 and FCE 24517, respectively. Isolated rat hearts perfused with concentrations of both derivatives equivalent to their respective IC50 values did not show any significant change in ECG parameters, contractility and coronary flow. Compared to control hearts, FCE 24517 10(-6) M induced a significant increase in PR interval, reduction in + dF/dtmax, heart rate and coronary flow, while FCE 24561 10(-6) M produced a modest but significant increase in S alpha T segment and decrease in + dF/dtmax. Rats treated with FCE 24561 3, 6 or 12 mg/kg, intravenously (i.v.), once weekly for 3 weeks had a modest increase in S alpha T segment and QRS complex duration, while a slight alteration of S alpha T segment and QRS complex duration were observed in rats given FCE 24517 1 or 2 mg/kg i.v. once weekly for 3 weeks. No cardiac histologic alterations were found in hearts from rats receiving FCE 24517 or FCE 24561. For comparison, the cardiotoxicity of doxorubicin (DXR) was evaluated in the same experimental models; perfusion of hearts with DXR 10(-6) M induced severe alterations in all parameters of the isolated hearts; the administration of DXR 3 mg/kg i.v. once a week for 3 weeks was associated with a widening of the S alpha T segment and QRS complex and cardiac histologic picture was markedly altered. In conclusion, distamycin A derivatives display elevated cytotoxicity while no substantial cardiotoxicity was observed.     

Patent Evaluation : WO9310130-: Istituto Nazionale per la Ricerca sul Cancro: Triamino platinum complexes and their potential as anticancer agents

Novelty: Triamino platinum complexes are claimed. They are stated to have in vivo antitumour activity without renal toxicity. They may be used to treat cancer.

Biology: Compounds had significant cytotoxicity in vitro against P388 murine leukaemic cells, K562 human erythroleukaemic cells, Jurkat human cells and cisplatin-resistant tumour cells. The preferred compound (cis-DPR) was evaluated in vitro on cisplatin-resistant L1210 murine leukaemic cell lines (24 hr thymidine-uptake method). IC₅₀ values were about 10 times superior to cisplatin. BDF1 mice infected with P388 leukaemic cells showed a significant increase in lifespan compared with cisplatin.

Chemistry: An example is provided for the preparation of cis-diamino-chloro-4-[diethylammoniumethoxycarbonylphenyl]amino-platinum (II) bis-chloride monohydrate (cis-DPR). This is the preferred compound used in the tests. Characterization is by mp, ir, uv and elemental analysis.     

Arzanol, a prenylated heterodimeric phloroglucinyl pyrone, inhibits eicosanoid biosynthesis and exhibits anti-inflammatory efficacy in vivo

Based on its capacity to inhibit in vitro HIV-1 replication in T cells and the release of pro-inflammatory cytokines in monocytes, the prenylated heterodimeric phloroglucinyl α-pyrone arzanol was identified as the major anti-inflammatory and anti-viral constituent from Helichrysum italicum. We have now investigated the activity of arzanol on the biosynthesis of pro-inflammatory eicosanoids, evaluating its anti-inflammatory efficacy in vitro and in vivo. Arzanol inhibited 5-lipoxygenase (EC 7.13.11.34) activity and related leukotriene formation in neutrophils, as well as the activity of cyclooxygenase (COX)-1 (EC 1.14.99.1) and the formation of COX-2-derived prostaglandin (PG)E₂in vitro (IC₅₀ = 2.3–9 μM). Detailed studies revealed that arzanol primarily inhibits microsomal PGE₂ synthase (mPGES)-1 (EC 5.3.99.3, IC₅₀ = 0.4 μM) rather than COX-2. In fact, arzanol could block COX-2/mPGES-1-mediated PGE₂ biosynthesis in lipopolysaccharide-stimulated human monocytes and human whole blood, but not the concomitant COX-2-derived biosynthesis of thromboxane B₂ or of 6-keto PGF_1α, and the expression of COX-2 or mPGES-1 protein was not affected. Arzanol potently suppressed the inflammatory response of the carrageenan-induced pleurisy in rats (3.6 mg/kg, i.p.), with significantly reduced levels of PGE₂ in the pleural exudates. Taken together, our data show that arzanol potently inhibits the biosynthesis of pro-inflammatory lipid mediators like PGE₂in vitro and in vivo, providing a mechanistic rationale for the anti-inflammatory activity of H. italicum, and a rationale for further pre-clinical evaluation of this novel anti-inflammatory lead.     

Immunosuppressive effects of new cyclolanostane triterpene diglycosides from the aerial part of <Emphasis Type="Italic">Cimicifuga foetida</Emphasis>

Two new cyclolanostane diglycosides, cimifoetiside A (1) and cimifoetiside B (2), were isolated from an 80% ethanolic extract of the aerial part of Cimicifuga foetida L. (Ranuculaceae). Using spectral data and chemical analysis, the structures of 1 and 2 were identified as (23R, 24S) cimicigenol 3-O-β-D-glucopyranosyl-(1″→3′)-β-D-xylopyranoside and (23R, 24S) cimicigenol 3-O-β-D-glucopyranosyl-(1″→2′)-β-D-xylopyranoside, respectively. The in vitro immunosuppressive effects of the two new compounds 1 and 2, as well as four other known cyclolanostane saponins 3–6 on T cells were evaluated. All the agents tested effectively inhibited the proliferation of murine splenocytes induced by Concanavalin A (ConA), with IC₅₀ values ranging from 12.7 nM to 33.3 nM.     

Pharmacologic and Pharmacokinetic Assessment of Anti-TGFβ2 Aptamers in Rabbit Plasma and Aqueous Humor

Purpose The aim of the study is to determine the bioactivity and effects of PEGylation on the pharmacokinetics in rabbit aqueous humor and plasma of an aptamer directed against TGFβ2. Methods Pharmacological activity of anti-TGFβ2 aptamer in rabbit ocular fluid was demonstrated using a mink lung epithelial cell proliferation assay. For pharmacokinetic analyses, concentrations of aptamers in plasma and aqueous humor were determined over time following bilateral subconjunctival administration to Dutch-belted rabbits using a hybridization-based pseudo-enzyme-linked immunosorbent assay (ELISA) assay. Results Anti-TGFβ2 aptamer (ARC81) binds to human TGFβ2 with a K_D of approximately 5 nM and inhibits the activity of human TGFβ2 in vitro in a cell-based assay with an IC₅₀ of approximately 100 nM. ARC81 blocks endogenously derived TGFβ2 in rabbit aqueous humor in vitro with an IC₅₀ of approximately 200 nM and an IC₉₀ of approximately 1 μM. In vivo in rabbit, ARC81 [no polyethylene glycol (PEG)] entered systemic circulation rapidly (t_max = 1 h in plasma) relative to aptamer conjugates ARC117 (20 kDa PEG) and ARC119 (40 kDa PEG), which showed prolonged residence in the subconjunctival space and aqueous compartment (t_max = 6 and 12 h, respectively, in plasma). Both 20- and 40-kDa aptamer conjugates reached maximal concentrations (C_max) in aqueous humor of 23–30 nM and remained at or above 1 nM for as long as 12 h. Conclusions Pharmacologically active levels of anti-TGFβ2 aptamers can be sustained in the ocular fluid and local tissue environment over a 12-h period after single administration. Daily subconjunctival administration of PEGylated anti-TGFβ2 aptamers should allow further pharmacological evaluation of these agents in a rabbit conjunctival scarring model. Perioperative administration, via subconjunctival injection, may prove to be an effective means to deliver therapeutic quantities of TGFβ2 aptamer conjugates in trabeculectomy procedures.     

Inhibition of P-glycoprotein in Caco-2 cells: Effects of herbal remedies frequently used by cancer patients

1. The herbal products Natto K2, Agaricus, mistletoe, noni juice, green tea and garlic were investigated for in vitro inhibitory potential on P-glycoprotein (P-gp)-mediated transport of digoxin (30 nM) in differentiated and polarized Caco-2 cells. 2.?Satisfactory cell functionality was demonstrated through measurements of assay linearity, transepithelial electric resistance (TEER), cytotoxicity, mannitol permeability, and inclusion of the positive inhibition control verapamil. 3.?The most potent inhibitors of the net digoxin flux (IC₅₀) were mistletoe?>?Natto K2?>?Agaricus?>?green tea (0.022, 0.62, 3.81, >4.5 mg ml^?1, respectively). Mistletoe also showed the lowest IC₂₅ value, close to that obtained by verapamil (1.0 and 0.5 µg ml^?1, respectively). The IC₅₀/IC₂₅ ratio was found to be a good parameter for the determination of inhibition profiles. Garlic and noni juice were classified as non-inhibitors. 4.?This study shows that mistletoe, Natto K2, Agaricus and green tea inhibit P-gp in vitro. Special attention should be paid to mistletoe due to very low IC₅₀ and IC₂₅ values and to Natto K2 due to a low IC₅₀ value and a low IC₅₀/IC₂₅ ratio.     

Quantitative projection of human brain penetration of the H3 antagonist PF-03654746 by integrating rat-derived brain partitioning and PET receptor occupancy

1.?Unbound brain drug concentration (C_b,u), a valid surrogate of interstitial fluid drug concentration (C_ISF), cannot be directly determined in humans, which limits accurately defining the human C_b,u:C_p,u of investigational molecules.

2.?For the H₃R antagonist (1R,3R)-N-ethyl-3-fluoro-3-[3-fluoro-4-(pyrrolidin-1-lmethyl)phenyl]cyclobutane-1-carboxamide (PF-03654746), we interrogated C_b,u:C_p,u in humans and nonhuman primate (NHP).

3.?In rat, PF-03654746 achieved net blood–brain barrier (BBB) equilibrium (C_b,u:C_p,u of 2.11).

4.?In NHP and humans, the PET receptor occupancy-based C_p,u IC₅₀ of PF-03654746 was 0.99?nM and 0.31?nM, respectively, which were 2.1- and 7.4-fold lower than its in vitro human H₃ K_i (2.3?nM).

5.?In an attempt to understand this higher-than-expected potency in humans and NHP, rat-derived C_b,u:C_p,u of PF-03654746 was integrated with C_p,u IC₅₀ to identify unbound (neuro) potency of PF-03654746, nIC₅₀.

6.?The nIC₅₀ of PF-03654746 was 2.1?nM in NHP and 0.66?nM in human which better correlated (1.1- and 3.49-fold lower) with in vitro human H₃ K_i (2.3?nM).

7.?This correlation of the nIC₅₀ and in vitro hH₃ K_i suggested the translation of net BBB equilibrium of PF-03654746 from rat to NHP and humans, and confirmed the use of C_p,u as a reliable surrogate of C_b,u.

8.?Thus, nIC₅₀ quantitatively informed the human C_b,u:C_p,u of PF-03654746.     

Effects of aripiprazole and its active metabolite dehydroaripiprazole on the activities of drug efflux transporters expressed both in the intestine and at the blood-brain barrier

The inhibition potencies of aripiprazole and its active metabolite, dehydroaripiprazole, on the activities of human multidrug resistance protein 1 (MDR1/ABCB1; P‐glycoprotein), breast cancer resistance protein (BCRP/ABCG2) and multidrug resistance‐associated protein 4 (MRP4/ABCC4), that are drug efflux transporters expressed both in the intestine and at the blood–brain barrier (BBB), were investigated. Aripiprazole and dehydroapripiprazole showed relatively strong inhibitory effects on human MDR1 with IC₅₀ values of 1.2 and 1.3 µm in human MDR1‐transfected Mardin‐Darby canine kidney (MDCKII‐MDR1) cells, respectively. The inhibition potencies of other atypical antipsychotics (risperidone, paliperidone, olanzapine and ziprasidone) for human MDR1 were also evaluated using the same in vitro experimental system and IC₅₀ values were more than 10‐fold higher than those of the two compounds. Aripiprazole and dehydroaripiprazole also had inhibition potencies against human BCRP with IC₅₀ values of 3.5 and 0.52 µm , respectively. The ratios of steady‐state unbound concentrations of aripiprazole and dehydroaripiprazole to their IC₅₀ values against human MDR1 and BCRP activities were less than 0.1, whereas the theoretically maximum gastrointestinal concentration of aripiprazole ([I]₂) to its IC₅₀ values was much higher than the cut‐off value of 10, proposed by the International Transporter Consortium (ITC) and the Food and Drug Administration (FDA). In contrast, aripiprazole and dehydroaripiprazole showed almost no inhibitory effect against the activity of human MRP4. These findings indicate that aripiprazole is unlikely to cause drug–drug interactions (DDIs) at the BBB when co‐administered with substrate drugs of these drug transporters investigated. However, interactions at the intestinal absorption process may be of concern. Copyright © 2012 John Wiley & Sons, Ltd.     

Potentiation of the antitumor effect of 11-keto-β-boswellic acid by its 3-α-hexanoyloxy derivative

We recently discovered that a propionyloxy derivative of 11-keto-β-boswellic acid (PKBA) showed better anticancer potential than other boswellic acids including AKBA, encompassing the importance of acyl group at the 3-α-hydroxy position of KBA. In continuation of our previous work, other higher derivatives (with increasing alkoxy chain length at 3-α-hydroxy position) including butyryloxy (BKBA) and hexanoyloxy (HKBA) derivatives of KBA were synthesized. The respective IC₅₀ values of BKBA and HKBA in HL-60 cells were found to be 7.7 and 4.5 μg/ml. IC₅₀ value of HKBA was comparatively lower than that of BKBA, and further lower than that of the previously reported derivative (PKBA, IC₅₀ 8.7 μg/ml). In order to compare the anticancer potential of HKBA with PKBA, detailed in vitro pro-apoptotic and in vivo anticancer studies were carried out. The induction of apoptosis by HKBA was measured using various parameters including fluorescence and scanning electron microscopy, DNA fragmentation and Annexin V-FITC binding. The extent of DNA damage was measured using neutral comet assay. HKBA was further evaluated for its effect on DNA cell cycle and mitochondria where it was found to arrest cells in G₂/M phase and also induced loss of mitochondrial membrane potential. These events were associated with increased expression of cytosolic cytochrome c and cleavage of PARP. Target based studies showed that HKBA inhibited the enzymatic activity of topoisomerases I and II at low doses than that of PKBA. In vivo studies also revealed a low dose inhibitory effect of HKBA on ascitic and solid murine tumor models.     

Selective toxicity of ginsenoside Rg<Subscript>3</Subscript> on multidrug resistant cells by membrane fluidity modulation

Multidrug resistance (MDR) is a major problem in cancer chemotherapy. It was previously reported that a red ginseng saponin, 20(S)-ginsenoside Rg₃ could modulate MDR in vitro and extend the survival of mice implanted with ADR-resistant murine leukemia P388 cells. This study examined the cytotoxicity of Rg₃ on normal and transformed cells, along with its effect on the membrane fluidity. The cytotoxicity study revealed that 120 μM of Rg₃ was cytotoxic against a multidrug-resistant human fibroblast carcinoma cell line, KB V20C, but not against normal WI 38 cells in vitro. Flow cytometric analysis using rhodamine 123 as the artificial substrate showed that Rg₃ promoted the accumulation of rhodamine 123 in ADR-resistant murine leukemia P388 cells in vivo. Fluorescence polarization studies using the hydrophilic fluorescent probe, DPH, and hydrophobic probe, TMA-DPH, showed that 20 μM Rg₃ induced a significant increase in fluorescence anisotropy in KB V20C cells but not in the parental KB cells. These results clearly show that Rg₃ decreases the membrane fluidity thereby blocking drug efflux. These two authors contributed equally to this work.     

Polychlorinated biphenyls disrupt hepatic epidermal growth factor receptor signaling

1.?Polychlorinated biphenyls (PCBs) are persistent environmental pollutants that disrupt hepatic xenobiotic and intermediary metabolism, leading to metabolic syndrome and nonalcoholic steatohepatitis (NASH).

2.?Since phenobarbital indirectly activates Constitutive Androstane Receptor (CAR) by antagonizing growth factor binding to the epidermal growth factor receptor (EGFR), we hypothesized that PCBs may also diminish EGFR signaling.

3.?The effects of the PCB mixture Aroclor 1260 on the protein phosphorylation cascade triggered by EGFR activation were determined in murine (in vitro and in vivo) and human models (in vitro). EGFR tyrosine residue phosphorylation was decreased by PCBs in all models tested.

4.?The IC₅₀ values for Aroclor 1260 concentrations that decreased Y1173 phosphorylation of EGFR were similar in murine AML-12 and human HepG2 cells (～2–4?μg/mL). Both dioxin and non-dioxin-like PCB congeners decreased EGFR phosphorylation in cell culture.

5.?PCB treatment reduced phosphorylation of downstream EGFR effectors including Akt and mTOR, as well as other phosphoprotein targets including STAT3 and c-RAF in vivo.

6.?PCBs diminish EGFR signaling in human and murine hepatocyte models and may dysregulate critical phosphoprotein regulators of energy metabolism and nutrition, providing a new mechanism of action in environmental diseases.     

Isolation of a multidrug resistance inhibitor fromAconitum pseudo-laeve var.erectum

To overcome multidrug resistance (MDR) in cancer chemotherapy, we prepared various plant extracts and searched for a component which is effective for inhibition of MDR. MDR inhibition activity was determined by measuring cytotoxicity to MDR cells using multidrug resistant human fibrocarcinoma KB V20C, which is resistant to 20 nM vincristine and expresses high level ofmdr1 gene. Of various plant extracts, the MeOH extract of the root ofAconitum pseudo-laeve var.erectum was found to have potent inhibitory activity on MDR. The bioassay-guided fractionation of the MeOH extract of the plant led to the isolation of an alkaloid, lycaconitine, as an active principle. And the IC₅₀ of lycaconitne for KB V20C cells was 74 μg/ml.     

Leishmanicidal and cytotoxic activities of Nigella sativa and its active principle,thymoquinone

Abstract

Context: Leishmaniasis is a complex disease with a broad spectrum of clinical presentations.

Objective: We evaluated the anti-leishmanial effects of Nigella sativa L. (Ranunculaceae) against Leishmania tropica and Leishmania infantum with an in vitro model.

Materials and methods: Antileishmanial effects of essential oil and methanolic extract of N. sativa (0–200?µg/mL) and thymoquinone (0–25?µg/mL) on promastigotes of both species and their cytotoxicity activities against murine macrophages were evaluated using the MTT assay at 24, 48, and 72?h. Moreover, their leishmanicidal effects against amastigotes were investigated in a macrophage model, for 48 and 72?h.

Results: The findings showed that essential oil (L. tropica IC₅₀ 9.3?μg/mL and L. infantum IC₅₀ 11.7?μg/mL) and methanolic extract (L. tropica IC₅₀ 14.8?μg/mL and L. infantum IC₅₀ 15.7?μg/mL) of N. sativa, particularly thymoquinone (L. tropica IC₅₀ 1.16?μg/mL and L. infantum IC₅₀ 1.47?μg/mL), had potent antileishmanial activity on promastigotes of both species after 72?h. In addition, essential oil (L. tropica IC₅₀ 21.4?μg/mL and L. infantum IC₅₀ 26.3?μg/mL), methanolic extract (L. tropica IC₅₀ 30.8?μg/mL and L. infantum IC₅₀ 34.6?μg/mL), and thymoquinone (L. tropica IC₅₀ 2.1?μg/mL and L. infantum IC₅₀ 2.6?μg/mL) mediated a significant decrease in the growth rate of amastigote forms of both species. Thymoquinone (CC₅₀ 38.8?μg/mL) exhibited higher cytotoxic effects against murine macrophages than the other extracts.

Conclusion: N. sativa, especially its active principle, thymoquinone, showed a potent leishmanicidal activity against L. tropica and L.infantum with an in vitro model.