Anti-angiogenic and vascular disrupting effects of C9, a new microtubule-depolymerizing agent

Background and purpose:

The critical role of blood supply in the growth of solid tumours makes blood vessels an ideal target for anti-tumour drug discovery. The anti-angiogenic and vascular disrupting activities of C9, a newly synthesized microtubule-depolymerizing agent, were investigated with several in vitro and in vivo models. Possible mechanisms involved in its activity were also assessed.

Experimental approach:

Microtubule-depolymerizing actions were assessed by surface plasmon resonance binding, competitive inhibition and cytoskeleton immunofluorescence. Anti-angiogenic and vascular disrupting activities were tested on proliferation, migration, tube formation with human umbilical vein endothelial cells, and in rat aortic ring, chick chorioallantoic membrane and Matrigel plug assays. Western blots and Rho activation assays were employed to examine the role of Raf-MEK-ERK (mitogen-activated ERK kinase, extracellular signal-regulated kinase) and Rho/Rho kinase signalling.

Key results:

C9 inhibited proliferation, migration and tube formation of endothelial cells and inhibited angiogenesis in aortic ring and chick chorioallantoic membrane assays. C9 induced disassembly of microtubules in endothelial cells and down-regulated Raf-MEK-ERK signalling activated by pro-angiogenic factors. In addition, C9 disrupted capillary-like networks and newly formed vessels in vitro and rapidly decreased perfusion of neovasculature in vivo. Endothelial cell contraction and membrane blebbing induced by C9 in neovasculature was dependent on the Rho/Rho kinase pathway.

Conclusions and implications:

Anti-angiogenic and vascular disruption by C9 was associated with changes in morphology and function of endothelial cells, involving the Raf-MEK-ERK and Rho/Rho kinase signalling pathways. These findings strongly suggest that C9 is a new microtubule-binding agent that could effectively target tumour vasculature.     

WTC-01, a novel synthetic oxime-flavone compound,destabilizes microtubules in human nasopharyngeal carcinoma cells in vitro and in vivo

Background and Purpose

Dynamic polymerization of microtubules is essential for cancer cell growth and metastasis, and microtubule-disrupting agents have become the most successful anti-cancer agents in clinical use. Besides their antioxidant properties, flavonoids also exhibit strong microtubule-disrupting activity and inhibit tumour growth. We have designed, synthesized and tested a series of oxime/amide-containing flavone derivatives. Here we report the evaluation of one compound, WTC-01 for its anti-proliferative effects in human cancer cells.

Experimental approach

We used a range of cancer cell lines including two human nasopharyngeal carcinoma (NPC) cell lines, measuring proliferation, cell cycle and apoptosis, along with caspase levels and mitochondrial membrane potentials. Assays of tubulin polymerisation in vitro and computer modelling of the colchicine binding site in tubulin were also used. In mice, pharmacokinetics and growth of NPC-derived tumours were studied.

Key Results

WTC-01 was most potent against proliferation of NPC cells (IC₅₀ = 0.45 μM), inducing accumulation of cells in G₂/M and increasing apoptosis, time- and concentration-dependently. The colchicine competition-binding experiments and computer modelling results suggested that WTC-01 causes microtubule disruption via binding to the colchicine-binding site of tubulin resulting in mitochondrial membrane damage and cell apoptosis via activation of caspase-9/-3 without noticeable activation of the caspase-8. Notably, our in vivo studies demonstrated that at doses of 25 and 50 mg·kg⁻¹, WTC-01 exhibited good pharmacokinetic properties and completely inhibited the growth of NPC-TW01 cells in a xenograft nude mouse model.

Conclusions and Implications

WTC-01, a new synthetic oxime-containing flavone, exhibited potent anti-tumour activity against NPC cells and merits further investigation.     

Hydrogen sulphide-releasing diclofenac derivatives inhibit breast cancer-induced osteoclastogenesis in vitro and prevent osteolysis ex vivo

BACKGROUND AND PURPOSE

Hydrogen sulphide (H₂S) and prostaglandins are both involved in inflammation, cancer and bone turnover, and non-steroidal anti-inflammatory drugs (NSAIDs) and H₂S donors exhibit anti-inflammatory and anti-tumour properties. H₂S-releasing diclofenac (S-DCF) derivatives are a novel class of NSAIDs combining the properties of a H₂S donor with those of a conventional NSAID.

EXPERIMENTAL APPROACH

We studied the effects of the S-DCF derivatives ACS15 and ACS32 on osteoclast and osteoblast differentiation and activity in vitro, human and mouse breast cancer cells support for osteoclast formation and signalling in vitro, and osteolysis ex vivo.

KEY RESULTS

The S-diclofenac derivatives ACS15 and ACS32 inhibited the increase in osteoclast formation induced by human MDA-MB-231 and MCF-7 and mouse 4T1 breast cancer cells without affecting breast cancer cell viability. Conditioned media from human MDA-MB-231 cells enhanced IκB phosphorylation and osteoclast formation and these effects were significantly inhibited following treatment by ACS15 and ACS32, whereas the parent compound diclofenac had no effects. ACS15 and ACS32 inhibited receptor activator of NFκB ligand-induced osteoclast formation and resorption, and caused caspase-3 activation and apoptosis in mature osteoclasts via a mechanism dependent on IKK/NFκB inhibition. In calvaria organ culture, human MDA-MB-231 cells caused osteolysis, and this effect was completely prevented following treatment with ACS15 and ACS32.

CONCLUSIONS AND IMPLICATIONS

S-diclofenac derivatives inhibit osteoclast formation and activity, suppress breast cancer cell support for osteoclastogenesis and prevent osteolysis. This suggests that H₂S-releasing diclofenac derivatives exhibit anti-resorptive properties, which might be of clinical value in the treatment of osteolytic bone disease.     

Anti-tumour effects of small interfering RNA targeting anion exchanger 1 in experimental gastric cancer

BACKGROUND AND PURPOSE

Anion exchanger 1 (AE1) is an integral membrane protein found in erythrocytes. Our previous studies have demonstrated that AE1 is expressed in human gastric cancer cells and may be involved in the carcinogenesis of cancer. In this study, we further investigated the role of AE1 in gastric carcinogenesis and the anti-tumour effects of AE1-targeted small interfering RNAs (siRNAs) in two experimental models of gastric cancer.

EXPERIMENTAL APPROACH

Molecular and cellular experiments were performed to elucidate the role of AE1 in the malignant transformation of gastric epithelium and the effects of AE1-targeted siRNAs on gastric cancer cells. The anti-tumour effect of the siRNA was evaluated in vivo in two mouse models, nude mice implanted with human gastric cancer xenografts (Model I) and mice with gastric cancer induced by N-methyl-N-nitrosourea (MNU) and Helicobacter pylori (Model II).

KEY RESULTS

AE1 was found to increase gastric carcinogenesis by promoting cell proliferation. AE1-targeted siRNA significantly suppressed AE1 expression and hindered tumour growth. Furthermore, the siRNA markedly decreased the detection rate of gastric cancer, in parallel with an increase in atypical hyperplasia at the end of the experiment in Model II.

CONCLUSIONS AND IMPLICATIONS

Knockdown of AE1 expression in gastric mucosa by administration of synthetic siRNAs significantly inhibits the growth of gastric cancer and decreases the detection rate of this tumour in experimental mice. These results suggest that AE1 is potentially a key therapeutic target and the silencing of AE1 expression in gastric mucosa could provide a new therapeutic approach for treating gastric cancer.     

Enhanced anti-tumour effects of Vinca alkaloids given separately from cytostatic therapies

Background and Purpose

In polychemotherapy protocols, that is for treatment of neuroblastoma and Ewing sarcoma, Vinca alkaloids and cell cycle-arresting drugs are usually administered on the same day. Here we studied whether this combination enables the optimal antitumour effects of Vinca alkaloids to be manifested.

Experimental Approach

Vinca alkaloids were tested in a preclinical mouse model in vivo and in vitro in combination with cell cycle-arresting drugs. Signalling pathways were characterized using RNA interference.

Key Results

In vitro, knockdown of cyclins significantly inhibited vincristine-induced cell death indicating, in accordance with previous findings, Vinca alkaloids require active cell cycling and M-phase transition for induction of cell death. In contrast, anthracyclines, irradiation and dexamethasone arrested the cell cycle and acted like cytostatic drugs. The combination of Vinca alkaloids with cytostatic therapeutics resulted in diminished cell death in 31 of 36 (86%) tumour cell lines. In a preclinical tumour model, anthracyclines significantly inhibited the antitumour effect of Vinca alkaloids in vivo. Antitumour effects of Vinca alkaloids in the presence of cytostatic drugs were restored by caffeine, which maintained active cell cycling, or by knockdown of p53, which prevented drug-induced cell cycle arrest. Therapeutically most important, optimal antitumour effects were obtained in vivo upon separating the application of Vinca alkaloids from cytostatic therapeutics.

Conclusion and Implications

Clinical trials are required to prove whether Vinca alkaloids act more efficiently in cancer patients if they are applied uncoupled from cytostatic therapies. On a conceptual level, our data suggest the implementation of polychemotherapy protocols based on molecular mechanisms of drug–drug interactions.

Linked Article

This article is commented on by Solary, pp 1555–1557 of this issue. To view this commentary visit http://dx.doi.org/10.1111/bph.12101     

SZ-685C,a marine anthraquinone,is a potent inducer of apoptosis with anticancer activity by suppression of the Akt/FOXO pathway

Background and purpose:

The aims of this study were to investigate the anti-cancer activity of SZ-685C, an anthracycline analogue isolated from marine-derived mangrove endophytic fungi, and to explore the molecular mechanisms underlying such activity.

Experimental approach:

The effect of SZ-685C on the viability of cancer cell lines was investigated using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) assay. SZ-685C-induced apoptosis was assessed by Annexin V-fluorescein isothiocyanate/propidium iodide staining, terminal deoxynucleotidyl transferase-mediated dUTP nick end labelling assay and analysis of caspase activation. The effect of SZ-685C on the Akt/FOXO pathway was studied using Western blotting analysis, and the in vivo anti-tumour efficacy was examined in an MDA-MB-435 breast cancer xenograft model.

Key results:

SZ-685C suppressed the proliferation of six cancer cell lines derived from human breast cancer, prostate cancer, glioma and hepatoma (IC₅₀ values ranged from 3.0 to 9.6 µM) and the growth of breast cancer xenografts in mice. SZ-685C had a direct apoptosis-inducing effect through both the extrinsic and intrinsic apoptotic pathways, as shown by activation of caspase-8 and 9 as well as effector caspase-3 and poly (ADP-ribose) polymerase. Phosphorylation of Akt and its downstream effectors, forkhead box protein O1 and forkhead box protein O3a, was down-regulated in SZ-685C-treated cancer cells. Furthermore, the pro-apoptotic protein Bim was up-regulated by SZ-685C treatment consistent with FOXO dephosphorylation.

Conclusions and implications:

SZ-685C could induce apoptosis through the Akt/FOXO pathway, which consequently leads to the observed anti-tumour effect both in vitro and in vivo. Our data suggest that SZ-685C may be a potentially promising Akt inhibitor and anti-cancer drug candidate.     

PM01183, a new DNA minor groove covalent binder with potent in vitro and in vivo anti-tumour activity

BACKGROUND AND PURPOSE

PM01183 is a new synthetic tetrahydroisoquinoline alkaloid that is currently in phase I clinical development for the treatment of solid tumours. In this study we have characterized the interactions of PM01183 with selected DNA molecules of defined sequence and its in vitro and in vivo cytotoxicity.

EXPERIMENTAL APPROACH

DNA binding characteristics of PM01183 were studied using electrophoretic mobility shift assays, fluorescence-based melting kinetic experiments and computational modelling methods. Its mechanism of action was investigated using flow cytometry, Western blot analysis and fluorescent microscopy. In vitro anti-tumour activity was determined by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay and the in vivo activity utilized several human cancer models.

KEY RESULTS

Electrophoretic mobility shift assays demonstrated that PM01183 bound to DNA. Fluorescence-based thermal denaturation experiments showed that the most favourable DNA triplets providing a central guanine for covalent adduct formation are AGC, CGG, AGG and TGG. These binding preferences could be rationalized using molecular modelling. PM01183–DNA adducts in living cells give rise to double-strand breaks, triggering S-phase accumulation and apoptosis. The potent cytotoxic activity of PM01183 was ascertained in a 23-cell line panel with a mean GI₅₀ value of 2.7 nM. In four murine xenograft models of human cancer, PM01183 inhibited tumour growth significantly with no weight loss of treated animals.

CONCLUSIONS AND IMPLICATIONS

PM01183 is shown to bind to selected DNA sequences and promoted apoptosis by inducing double-strand breaks at nanomolar concentrations. The potent anti-tumour activity of PM01183 in several murine models of human cancer supports its development as a novel anti-neoplastic agent.     

JNK is required for maintaining the tumor-initiating cell-like properties of acquired chemoresistant human cancer cells

Aim:

Many studies reveal an association between the acquired chemoresistant phenotype of cancer cells and tumor-initiating cell-like properties. The aim of this study was to determine the impact of c-Jun N-terminal kinase (JNK) on the tumor-initiating cell-like properties of acquired chemoresistant human cancer cells.

Methods:

Two well-established human acquired chemoresistant cancer cell lines K562/A02 and KB/VCR, as well as their respective parental counterparts K562 and KB were tested. The expression of relevant mRNAs and proteins was detected using qRT-PCR and Western blotting, respectively. Sphere formation and self-renewal assays were used to study the tumor-initiating cell-like properties. Soft agar and colony formation assays were used to investigate tumorigenic ability.

Results:

We observed that suppressing JNK activity by its specific small molecule inhibitor SP600125 or by limiting JNK1/2 expression with JNK1/2 shRNA lentiviruses inhibited the expression of pluripotent stem cell markers such as Oct4, Sox2, and Nanog in KB/VCR cells and K562/A02 cells as well as sphere formation and self-renewal abilities of K562/A02 cells. Additionally, inhibition of JNK activity significantly inhibited the in vitro and in vivo tumor-initiating abilities of KB/VCR cells. Furthermore, our data suggest that blocking JNK activity abundantly inhibited the Hedgehog (Hh) pathway activity, as reflected by reduction of Hedgehog (Hh) pathway target genes Gli1 and ptch1 at the mRNA level as well as Gli-luciferase activity.

Conclusion:

JNK maintains the tumor-initiating cell-like properties of acquired chemoresistant K562/A02 and KB/VCR cells potentially through activating the Hedgehog pathway. Thus, disruption of tumor-initiating cell-like properties by targeting JNK may be a new approach to combating acquired chemoresistance.     

Cucurbitacin B, a novel in vivo potentiator of gemcitabine with low toxicity in the treatment of pancreatic cancer

Background and purpose:

Pancreatic cancer is a highly aggressive malignancy, and improvement in systemic therapy is necessary to treat this frequently encountered metastatic disease. The current targeted agents used in combination with gemcitabine improved objective response rates, but with little or no improvements in survival and also increased toxicities in pancreatic cancer patients. Recently, we showed that the triterpenoid cucurbitacin B inhibited tumour growth in pancreatic cancer cells by inhibition of the JAK/STAT pathway, and synergistically increased antiproliferative effects of gemcitabine in vitro.

Experimental approach:

The anti-tumour effects and toxicities of cucurbitacin B in combination with gemcitabine were tested against human pancreatic cancer cells in a murine xenograft model.

Key results:

Combined therapy with cucurbitacin B and gemcitabine at relatively low doses (0.5 mg·kg⁻¹ and 25 mg·kg⁻¹ respectively) resulted in highly significant tumour growth inhibition of pancreatic cancer xenografts (up to 79%). Remarkably, this therapy was well tolerated by the animals, as shown by histology of visceral organs, analysis of serum chemistry, full blood counts and bone marrow colony numbers. Western blot analysis of the tumour samples of mice who received both cucurbitacin B and gemcitabine, revealed stronger inhibition of Bcl-XL, Bcl-2 and c-myc, and higher activation of the caspase cascades, than mice treated with either agent alone.

Conclusions and implications:

Combination of cucurbitacin B and gemcitabine had profound anti-proliferative effects in vivo against xenografts of human pancreatic cancer cells, without any significant signs of toxicity. This promising combination should be examined in therapeutic trials of pancreatic cancer.     

Phospho-ibuprofen (MDC-917) incorporated in nanocarriers: anti-cancer activity in vitro and in vivo

BACKGROUND AND PURPOSE

Phospho-ibuprofen (P-I; MDC-917) inhibits the growth of colon cancer in mice. Here, we investigated the use of nanocarriers to improve its pharmacokinetics (PKs) and anti tumour efficacy.

EXPERIMENTAL APPROACH

The cellular uptake and cytotoxicity of P-I encapsulated into liposomes and micelles, and its in vitro metabolic stability, were determined in cultures of human colon adenocarcinoma cells. The performance of liposomal P-I was further evaluated in PK studies in mice, and in a model of colon cancer xenografts in nude mice.

KEY RESULTS

Liposomal P-I and micellar P-I showed significantly enhanced cellular uptake in the colon cancer cells. Liposomal P-I also demonstrated increased cytotoxicity in vitro. Free P-I was metabolized rapidly to ibuprofen in the presence of purified esterases. In contrast, liposomal P-I, and to a lesser extent micellar P-I, was resistant to esterase-mediated hydrolysis. In mice, liposomal P-I partially protected P-I from hydrolysis in the circulation, and improved the biodistribution of intact P-I and its metabolites compared to free P-I. Liposomal P-I was more effective at inhibiting the growth of human colon cancer xenografts in mice, which may be explained on the basis of its improved PK profile compared to free P-I.

CONCLUSIONS AND IMPLICATIONS

Liposome encapsulation of P-I partially protected P-I from esterase-mediated hydrolysis in mice, enhanced the cytotoxicity and bioavailability of P-I and increased its efficacy at inhibiting the growth of human colon cancer xenografts. These results indicate that liposomes are suitable nanocarriers for the delivery of P-I, and that the anti-tumour potential of liposomal P-I merits further evaluation.     

Bp44mT: an orally active iron chelator of the thiosemicarbazone class with potent anti-tumour efficacy

BACKGROUND AND PURPOSE

Our previous studies demonstrated that a thiosemicarbazone iron chelator (di-2-pyridylketone-4,4-dimethyl-3-thiosemicarbazone; Dp44mT) possesses potent and selective anti-cancer activity but led to cardiotoxicity at non-optimal doses. In this study, we examined the in vivo anti-tumour efficacy and tolerability of a new-generation 2-benzoylpyridine thiosemicarbazone iron chelator (2-benzoylpyridine-4,4-dimethyl-3-thiosemicarbazone; Bp44mT) administered via the oral or i.v. routes.

EXPERIMENTAL APPROACH

BpT chelators were tested in vitro against human lung cancer cells (DMS-53) and in vivo in DMS-53 tumour xenografts in mice. The toxicity of Bp44mT in vivo and its effects on the expression of iron-regulated molecules involved in growth and cell cycle control were investigated.

KEY RESULTS

Administration of Bp44mT by either route resulted in marked dose-dependent inhibition of tumour growth. When administered at 50 mg·kg⁻¹ via oral gavage three times per week for 23 days, the net xenograft growth was inhibited by 75%, compared with vehicle-treated mice. Toxicological examination showed reversible alterations including slight reduction of RBC count, with a decrease of liver and splenic iron levels, which confirmed iron chelation in vivo. Importantly, in contrast to Dp44mT, the chelator-treated mice did not show cardiac histological abnormalities. There was also no significant weight loss in mice, suggesting oral administration of Bp44mT was well tolerated.

CONCLUSIONS AND IMPLICATIONS

This is the first study to show that Bp44mT can be given orally with potent anti-tumour efficacy. Oral administration of a novel and effective chemotherapeutic agent provides the benefits of convenience for chronic dosing regimens.     

The GPR 55 agonist,L-α-lysophosphatidylinositol,mediates ovarian carcinoma cell-induced angiogenesis

Background and Purpose

Highly vascularized ovarian carcinoma secretes the putative endocannabinoid and GPR55 agonist, L-α-lysophosphatidylinositol (LPI), into the circulation. We aimed to assess the involvement of this agonist and its receptor in ovarian cancer angiogenesis.

Experimental Approach

Secretion of LPI by three ovarian cancer cell lines (OVCAR-3, OVCAR-5 and COV-362) was tested by mass spectrometry. Involvement of cancer cell-derived LPI on angiogenesis was tested in the in vivo chicken chorioallantoic membrane (CAM) assay along with the assessment of the effect of LPI on proliferation, network formation, and migration of neonatal and adult human endothelial colony-forming cells (ECFCs). Engagement of GPR55 was verified by using its pharmacological inhibitor CID16020046 and diminution of GPR55 expression by four different target-specific siRNAs. To study underlying signal transduction, Western blot analysis was performed.

Key Results

Ovarian carcinoma cell-derived LPI stimulated angiogenesis in the CAM assay. Applied LPI stimulated proliferation, network formation, and migration of neonatal ECFCs in vitro and angiogenesis in the in vivo CAM. The pharmacological GPR55 inhibitor CID16020046 inhibited LPI-stimulated ECFC proliferation, network formation and migration in vitro as well as ovarian carcinoma cell- and LPI-induced angiogenesis in vivo. Four target-specific siRNAs against GPR55 prevented these effects of LPI on angiogenesis. These pro-angiogenic effects of LPI were transduced by GPR55-dependent phosphorylation of ERK1/2 and p38 kinase.

Conclusions and Implications

We conclude that inhibiting the pro-angiogenic LPI/GPR55 pathway appears a promising target against angiogenesis in ovarian carcinoma.     

Down-regulation of the Notch pathway mediated by a γ-secretase inhibitor induces anti-tumour effects in mouse models of T-cell leukaemia

Background and purpose:

γ-Secretase inhibitors (GSIs) block NOTCH receptor cleavage and pathway activation and have been under clinical evaluation for the treatment of malignancies such as T-cell acute lymphoblastic leukaemia (T-ALL). The ability of GSIs to decrease T-ALL cell viability in vitro is a slow process requiring >8 days, however, such treatment durations are not well tolerated in vivo. Here we study GSI''s effect on tumour and normal cellular processes to optimize dosing regimens for anti-tumour efficacy.

Experimental approach:

Inhibition of the Notch pathway in mouse intestinal epithelium was used to evaluate the effect of GSIs and guide the design of dosing regimens for xenograft models. Serum Aβ₄₀ and Notch target gene modulation in tumours were used to evaluate the degree and duration of target inhibition. Pharmacokinetic and pharmacodynamic correlations with biochemical, immunohistochemical and profiling data were used to demonstrate GSI mechanism of action in xenograft tumours.

Key results:

Three days of >70% Notch pathway inhibition was sufficient to provide an anti-tumour effect and was well tolerated. GSI-induced conversion of mouse epithelial cells to a secretory lineage was time- and dose-dependent. Anti-tumour efficacy was associated with cell cycle arrest and apoptosis that was in part due to Notch-dependent regulation of mitochondrial homeostasis.

Conclusions and implications:

Intermittent but potent inhibition of Notch signalling is sufficient for anti-tumour efficacy in these T-ALL models. These findings provide support for the use of GSI in Notch-dependent malignancies and that clinical benefits may be derived from transient but potent inhibition of Notch.     

Efficacy of a Non-Hormonal Treatment,BRN-01, on Menopausal Hot Flashes: A Multicenter,Randomized, Double-Blind,Placebo-Controlled Trial

Background

Homeopathic medicines have a place among the non-hormonal therapies for the treatment of hot flashes during the menopause.

Objective

The objective of this study was to evaluate the efficacy of the non-hormonal treatment BRN-01 in reducing hot flashes in menopausal women.

Study Design

This was a multicenter, randomized, double-blind, placebo-controlled study carried out between June 2010 and July 2011.

Setting

The study was conducted in 35 active centers in France (gynecologists in private practice).

Patients

One hundred and eight menopausal women, ≥50 years of age, were enrolled in the study. The eligibility criteria included menopause for <24 months and ≥5 hot flashes per day with a significant negative effect on the women’s professional and/or personal life.

Intervention

Treatment was either BRN-01 tablets, a registered homeopathic medicine containing Actaea racemosa (4 centesimal dilutions [4CH]), Arnica montana (4CH), Glonoinum (4CH), Lachesis mutus (5CH), and Sanguinaria canadensis (4CH), or identical placebo tablets, prepared by Laboratoires Boiron according to European Pharmacopoeia standards. Oral treatment (2 to 4 tablets per day) was started on day 3 after study enrollment and was continued for 12 weeks.

Main Outcome Measure

The main outcome measure was the hot flash score (HFS) compared before, during, and after treatment. Secondary outcome criteria were the quality of life (QoL) [measured using the Hot Flash Related Daily Interference Scale (HFRDIS)], severity of symptoms (measured using the Menopause Rating Scale), evolution of the mean dosage, and compliance. All adverse events (AEs) were recorded.

Results

One hundred and one women were included in the final analysis (intent-to-treat population: BRN-01, n = 50; placebo, n = 51). The global HFS over the 12 weeks, assessed as the area under the curve (AUC) adjusted for baseline values, was significantly lower in the BRN-01 group than in the placebo group (mean ± SD 88.2 ± 6.5 versus 107.2 ± 6.4; p = 0.0411). BRN-01 was well tolerated; the frequency of AEs was similar in the two treatment groups, and no serious AEs were attributable to BRN-01.

Conclusion

BRN-01 seemed to have a significant effect on the HFS, compared with placebo. According to the results of this clinical trial, BRN-01 may be considered a new therapeutic option with a safe profile for hot flashes in menopausal women who do not want or are not able to take hormone replacement therapy or other recognized treatments for this indication.Trial registration number (EudraCT): 2009-016959–21.     

Solid lipid nanoparticles of cholesteryl butyrate inhibit the proliferation of cancer cells in vitro and in vivo models

BACKGROUND AND PURPOSE

Solid lipid nanoparticles containing cholesteryl butyrate (cholbut SLN) can be a delivery system for the anti-cancer drug butyrate. These nanoparticles inhibit adhesion of polymorphonuclear and tumour cells to endothelial cells and migration of tumour cells, suggesting that they may act as anti-inflammatory and anti-tumour agents. Here we have evaluated the effects of cholbut SLN on tumour cell growth using in vitro and in vivo models.

EXPERIMENTAL APPROACH

Cholbut SLNs were incubated with cultures of four tumour cell lines, and cell growth was analysed by assessing viability, clonogenic capacity and cell cycle. Effects on intracellular signalling was assessed by Western blot analysis of Akt expression. The in vivo anti-tumour activity was measured in two models of PC-3 cell xenografts in SCID/Beige mice.

KEY RESULTS

Cholbut SLN inhibited tumour cell line viability, clonogenic activity, Akt phosphorylation and cell cycle progression. In mice injected i.v. with PC3-Luc cells and treated with cholbut SLN, . in vivo optical imaging and histological analysis showed no metastases in the lungs of the treated mice. In another set of mice injected s.c. with PC-3 cells and treated with cholbut SLN when the tumour diameter reached 2 mm, analysis of the tumour dimensions showed that treatment with cholbut SLN substantially delayed tumour growth.

CONCLUSION AND IMPLICATIONS

Cholbut SLN were effective in inhibiting tumour growth in vitro and in vivo. These effects may involve, in part, inhibition of Akt phosphorylation, which adds another mechanism to the activity of this multipotent drug.     

The fruit extract of Berberis crataegina DC: exerts potent antioxidant activity and protects DNA integrity

Background

Dried fruits of Berberis crataegina (Berberidaceae) have been frequently consumed as food garniture in Turkish cuisine, while its fruit paste has been used to increase stamina and in particular to prevent from cardiovascular dysfunctions in Northeastern Black Sea region of Turkey. This study investigated this folkloric information in order to explain the claimed healing effects as well as to evaluate possible risks.

Methods

Total phenolic, flavonoid and proanthocyanidin contents and antioxidant capacity of the methanolic fruit extract were evaluated through several in vitro assays. The cytotoxic and genotoxic effects of B. crataegina fruit extract were also assessed in both cervical cancer cell line (HeLa) and human peripheral blood lymphocytes.

Results

The extract showed protective effects against ferric-induced oxidative stress and had a relatively good antioxidant activity. It also ameliorated the H₂O₂ mediated DNA damage in lymphocytes, suggesting the protective effect against oxidative DNA damage.

Conclusion

The methanolic extract of B. crataegina fruits may be a potential antioxidant nutrient and also may exert a protective role against lipid peroxidation as well as oxidative DNA damage.Keyword: Berberis crataegina DC, Folk medicine, Genotoxicity, Lipid peroxidation, Antioxidant     

In vitro induction of T cells that are resistant to A2 adenosine receptor-mediated immunosuppression

Background and purpose:

The increased levels of extracellular adenosine in inflamed tissues down-regulate activated immune cells via the A_2A adenosine receptor. This A_2A adenosine receptor-mediated immunosuppression is a disqualifying obstacle in cancer immunotherapy as it protects cancerous tissues from adoptively transferred anti-tumour T cells. The aim of this study was to test whether the negative selection of T cells will produce T cells that are resistant to inhibition by extracellular adenosine.

Experimental approach:

Cytotoxic T lymphocytes (CTL) were developed by mixed lymphocyte culture in the presence or absence of the adenosine receptor agonist 5′-N-ethylcarboxamidoadenosine (NECA). The sensitivity of CTL to adenosine analogues was characterized by cAMP induction, interferon-γ production and cytotoxicity.

Key results:

CTL that could proliferate even in the presence of NECA were less susceptible to inhibition by A_2A adenosine receptor agonists, as shown by a much smaller accumulation of cAMP and less inhibition of interferon-γ production compared with control CTL. The successful protocol to produce CTL that are both resistant to adenosine-mediated immunosuppression and maintain strong cytotoxicity and interferon-γ secretion required NECA to be added only during the expansion stage after the establishment of CTL. In contrast, the priming of resting T cells in the presence of NECA resulted in T cells with impaired effector functions.

Conclusions and implications:

Adenosine-resistant effector T cells were successfully obtained by exposure of activated T cells to NECA. These in vitro studies form the basis for future attempts to produce anti-tumour T cells that are more effective in adoptive immunotherapy.     

Metabolic map and bioactivation of the anti-tumour drug noscapine

BACKGROUND AND PURPOSE

Noscapine is a promising anti-tumour agent. The purpose of the present study was to describe the metabolic map and investigate the bioactivation of noscapine.

EXPERIMENTAL APPROACH

Ultra-performance liquid chromatography coupled with electrospray ionization quadrupole time-of-flight mass spectrometry-based metabolomics was used to analyse the in vitro incubation mixtures, urine and faeces samples from mice treated with noscapine. Recombinant drug-metabolizing enzymes were employed to identify those involved in noscapine metabolism. Hepatic GSH levels and serum biochemistry were also carried out to determine reactive metabolites of noscapine.

KEY RESULTS

Several novel phase I metabolites of noscapine were detected after oral gavage of mice, including an N-demethylated metabolite, two hydroxylated metabolites, one metabolite undergoing both demethylation and cleavage of the methylenedioxy group and a bis-demethylated metabolite. Additionally, several novel glucuronides were detected, and their structures were elucidated through MS/MS fragmentology. Recombinant enzymes screening showed the involvement of several cytochromes P450, flavin-containing mono-oxygenase 1 and the UDP-glucuronosyltransferases UGT1A1, UGT1A3, UGT1A9 and UGT2B7, in noscapine metabolism. In vitro glutathione trapping revealed the existence of an ortho-quinone reactive intermediate formed through further oxidation of a catechol metabolite. However, this bioactivation process of noscapine does not occur in vivo. Similar to this result, altered glutathione levels in liver and serum biochemistry revealed no evidence of hepatic damage, thus indicating that, at least in mice, noscapine does not induce hepatotoxicity through bioactivation.

CONCLUSIONS AND IMPLICATIONS

A comprehensive metabolic map and bioactivation evaluation provides important information for the development of noscapine as an anti-tumour drug.     

Comparison of the IKr blockers moxifloxacin, dofetilide and E-4031 in five screening models of pro-arrhythmia reveals lack of specificity of isolated cardiomyocytes

BACKGROUND AND PURPOSE

Drug development requires the testing of new chemical entities for adverse effects. For cardiac safety screening, improved assays are urgently needed. Isolated adult cardiomyocytes (CM) and human embryonic stem cell-derived cardiomyocytes (hESC-CM) could be used to identify pro-arrhythmic compounds. In the present study, five assays were employed to investigate their sensitivity and specificity for evaluating the pro-arrhythmic properties of I_Kr blockers, using moxifloxacin (safe compound) and dofetilide or E-4031 (unsafe compounds).

EXPERIMENTAL APPROACH

Assays included the anaesthetized remodelled chronic complete AV block (CAVB) dog, the anaesthetized methoxamine-sensitized unremodelled rabbit, multi-cellular hESC-CM clusters, isolated CM obtained from CAVB dogs and isolated CM obtained from the normal rabbit. Arrhythmic outcome was defined as Torsade de Pointes (TdP) in the animal models and early afterdepolarizations (EADs) in the cell models.

KEY RESULTS

At clinically relevant concentrations (5–12 µM), moxifloxacin was free of pro-arrhythmic properties in all assays with the exception of the isolated CM, in which 10 µM induced EADs in 35% of the CAVB CM and in 23% of the rabbit CM. At supra-therapeutic concentrations (≥100 µM), moxifloxacin was pro-arrhythmic in the isolated rabbit CM (33%), in the hESC-CM clusters (18%), and in the methoxamine rabbit (17%). Dofetilide and E-4031 induced EADs or TdP in all assays (50–83%), and the induction correlated with a significant increase in beat-to-beat variability of repolarization.

CONCLUSION AND IMPLICATIONS

Isolated cardiomyocytes lack specificity to discriminate between TdP liability of the I_Kr blocking drugs moxifloxacin and dofetilide or E4031.