Lesley Larsen

Curcumin and synthetic analogs induce reactive oxygen species and decreases specificity protein (Sp) transcription factors by targeting microRNAs

BackgroundCurcumin inhibits growth of several cancer cell lines, and studies in this laboratory in bladder and pancreatic cancer cells show that curcumin downregulates specificity protein (Sp) transcription factors Sp1, Sp3 and Sp4 and pro-oncogenic Sp-regulated genes. In this study, we investigated the anticancer activity of curcumin and several synthetic cyclohexanone and piperidine analogs in colon cancer cells.MethodsThe effects of curcumin and synthetic analogs on colon cancer cell proliferation and apoptosis were determined using standardized assays. The changes in Sp proteins and Sp-regulated gene products were analysed by western blots, and real time PCR was used to determine microRNA-27a (miR-27a), miR-20a, miR-17-5p and ZBTB10 and ZBTB4 mRNA expression.ResultsThe IC50 (half-maximal) values for growth inhibition (24 hr) of colon cancer cells by curcumin and synthetic cyclohexanone and piperidine analogs of curcumin varied from 10 μM for curcumin to 0.7 μM for the most active synthetic piperidine analog RL197, which was used along with curcumin as model agents in this study. Curcumin and RL197 inhibited RKO and SW480 colon cancer cell growth and induced apoptosis, and this was accompanied by downregulation of specificity protein (Sp) transcription factors Sp1, Sp3 and Sp4 and Sp-regulated genes including the epidermal growth factor receptor (EGFR), hepatocyte growth factor receptor (c-MET), survivin, bcl-2, cyclin D1 and NFκB (p65 and p50). Curcumin and RL197 also induced reactive oxygen species (ROS), and cotreatment with the antioxidant glutathione significantly attenuated curcumin- and RL197-induced growth inhibition and downregulation of Sp1, Sp3, Sp4 and Sp-regulated genes. The mechanism of curcumin-/RL197-induced repression of Sp transcription factors was ROS-dependent and due to induction of the Sp repressors ZBTB10 and ZBTB4 and downregulation of microRNAs (miR)-27a, miR-20a and miR-17-5p that regulate these repressors.ConclusionsThese results identify a new and highly potent curcumin derivative and demonstrate that in cells where curcumin and RL197 induce ROS, an important underlying mechanism of action involves perturbation of miR-ZBTB10/ZBTB4, resulting in the induction of these repressors which downregulate Sp transcription factors and Sp-regulated genes.

Synthesis and cytotoxic potential of heterocyclic cyclohexanone analogues of curcumin

A series of 18 heterocyclic cyclohexanone analogues of curcumin have been synthesised and screened for their activity in both adherent and non-adherent cancer cell models. Cytotoxicity towards MBA-MB-231 breast cancer cells, as well as ability to inhibit NF-kappaB transactivation in non-adherent K562 leukemia cells were investigated. Three of these analogues 3,5-bis(pyridine-4-yl)-1-methylpiperidin-4-one B1, 3,5-bis(3,4,5-trimethoxybenzylidene)-1-methylpiperidin-4-one B10, and 8-methyl-2,4-bis((pyridine-4-yl)methylene)-8-aza-bicyclo[3.2.1]octan-3-one C1 showed potent cytotoxicity towards MBA-MB-231, MDA-MB-468, and SkBr3 cell lines with EC50 values below 1 microM and inhibition of NF-kappaB activation below 7.5 microM. The lead drug candidate, B10, was also able to cause 43% of MDA-MB-231 cells to undergo apoptosis after 18 h. This level of activity warrants further investigation for the treatment of ER-negative breast cancer and/or chronic myelogenous leukemia as prototypical cellular models for solid and liquid tumors.

Mechanisms for the activity of heterocyclic cyclohexanone curcumin derivatives in estrogen receptor negative human breast cancer cell lines

SummaryEstrogen receptor (ER)-negative breast cancer is an aggressive form that currently requires more drug treatment options. Thus, we have further modified cyclohexanone derivatives of curcumin and examined them for cytotoxicity towards ER-negative human breast cancer cells. Two of the analogs screened elicited increased cytotoxic potency compared to curcumin and other previously studied derivatives. Specifically, 2,6-bis(pyridin-3-ylmethylene)-cyclohexanone (RL90) and 2,6-bis(pyridin-4-ylmethylene)-cyclohexanone (RL91) elicited EC50 values of 1.54 and 1.10xa0µM, respectively, in MDA-MB-231 cells and EC50 values of 0.51 and 0.23 in SKBr3 cells. All other new compounds examined were less potent than curcumin, which elicited EC50 values of 7.6 and 2.4xa0µM in MDA-MB-231 and SKBr3 cells, respectively. Mechanistic analyses demonstrated that RL90 and RL91 significantly induced G2/M-phase cell cycle arrest and apoptosis. RL90 and RL91 also modulated the expression of key cell signaling proteins, specifically, in SKBr3 cells, protein levels of Her-2, Akt, and NFκB were decreased in a time-dependent manner, while activity of stress kinases JNK1/2 and P38 MAPK were increased. Signaling events in MDA-MB-231 cells were differently implicated, as EGFR protein levels were decreased and activity of GSK-3β transiently decreased, while β-catenin protein level and activity of P38 MAPK, Akt, and JNK1/2 were transiently increased. In conclusion replacement of the phenyl group of cyclohexanone derived curcumin derivatives with heterocyclic rings forms a class of second-generation analogs that are more potent than both curcumin and other derivatives. These new derivatives provide a platform for the further development of drugs for the treatment of ER-negative breast cancer.

A ratiometric fluorescent probe for assessing mitochondrial phospholipid peroxidation within living cells.

Mitochondrial oxidative damage contributes to a wide range of pathologies, and lipid peroxidation of the mitochondrial inner membrane is a major component of this disruption. However, despite its importance, there are no methods to assess mitochondrial lipid peroxidation within cells specifically. To address this unmet need we have developed a ratiometric, fluorescent, mitochondria-targeted lipid peroxidation probe, MitoPerOx. This compound is derived from the C11-BODIPY(581/591) probe, which contains a boron dipyromethane difluoride (BODIPY) fluorophore conjugated via a dienyl link to a phenyl group. In response to lipid peroxidation the fluorescence emission maximum shifts from ∼590 to ∼520nm. To target this probe to the matrix-facing surface of the mitochondrial inner membrane we attached a triphenylphosphonium lipophilic cation, which leads to its selective uptake into mitochondria in cells, driven by the mitochondrial membrane potential. Here we report on the development and characterization of MitoPerOx. We found that MitoPerOx was taken up very rapidly into mitochondria within cells, where it responded to changes in mitochondrial lipid peroxidation that could be measured by fluorimetry, confocal microscopy, and epifluorescence live cell imaging. Importantly, the peroxidation-sensitive change in fluorescence at 520nm relative to that at 590nm enabled the use of the probe as a ratiometric fluorescent probe, greatly facilitating assessment of mitochondrial lipid peroxidation in cells.

Using exomarkers to assess mitochondrial reactive species in vivo

BACKGROUNDnThe ability to measure the concentrations of small damaging and signalling molecules such as reactive oxygen species (ROS) in vivo is essential to understanding their biological roles. While a range of methods can be applied to in vitro systems, measuring the levels and relative changes in reactive species in vivo is challenging.nnnSCOPE OF REVIEWnOne approach towards achieving this goal is the use of exomarkers. In this, exogenous probe compounds are administered to the intact organism and are then transformed by the reactive molecules in vivo to produce a diagnostic exomarker. The exomarker and the precursor probe can be analysed ex vivo to infer the identity and amounts of the reactive species present in vivo. This is akin to the measurement of biomarkers produced by the interaction of reactive species with endogenous biomolecules.nnnMAJOR CONCLUSIONS AND GENERAL SIGNIFICANCEnOur laboratories have developed mitochondria-targeted probes that generate exomarkers that can be analysed ex vivo by mass spectrometry to assess levels of reactive species within mitochondria in vivo. We have used one of these compounds, MitoB, to infer the levels of mitochondrial hydrogen peroxide within flies and mice. Here we describe the development of MitoB and expand on this example to discuss how better probes and exomarkers can be developed. This article is part of a Special Issue entitled Current methods to study reactive oxygen species - pros and cons and biophysics of membrane proteins. Guest Editor: Christine Winterbourn.

Application of an online post‐column derivatization HPLC‐DPPH assay to detect compounds responsible for antioxidant activity in Sonchus oleraceus L. leaf extracts

To use an online assay to identify key antioxidants in Sonchus oleraceus leaf extracts and to investigate the effect of leaf position and extraction conditions on antioxidant concentration and activity.

Synthesis and anti-inflammatory structure–activity relationships of thiazine–quinoline–quinones: Inhibitors of the neutrophil respiratory burst in a model of acute gouty arthritis

Sixteen new thiazine-quinoline-quinones have been synthesised, plus one bicyclic analogue. These compounds inhibited neutrophil superoxide production in vitro with IC(50)s as low 60 nM. Compounds with high in vitro anti-inflammatory activity were also tested in a mouse model of acute inflammation. The most active compounds inhibited both neutrophil infiltration and superoxide production at doses 2.5 micromol/kg, highlighting their potential for development as novel NSAIDs.

Re-Directing an Alkylating Agent to Mitochondria Alters Drug Target and Cell Death Mechanism

We have successfully delivered a reactive alkylating agent, chlorambucil (Cbl), to the mitochondria of mammalian cells. Here, we characterize the mechanism of cell death for mitochondria-targeted chlorambucil (mt-Cbl) in vitro and assess its efficacy in a xenograft mouse model of leukemia. Using a ρ° cell model, we show that mt-Cbl toxicity is not dependent on mitochondrial DNA damage. We also illustrate that re-targeting Cbl to mitochondria results in a shift in the cell death mechanism from apoptosis to necrosis, and that this behavior is a general feature of mitochondria-targeted Cbl. Despite the change in cell death mechanisms, we show that mt-Cbl is still effective in vivo and has an improved pharmacokinetic profile compared to the parent drug. These findings illustrate that mitochondrial rerouting changes the site of action of Cbl and also alters the cell death mechanism drastically without compromising in vivo efficacy. Thus, mitochondrial delivery allows the exploitation of Cbl as a promiscuous mitochondrial protein inhibitor with promising therapeutic potential.

A mitochondria-targeted mass spectrometry probe to detect glyoxals: implications for diabetes

The glycation of protein and nucleic acids that occurs as a consequence of hyperglycemia disrupts cell function and contributes to many pathologies, including those associated with diabetes and aging. Intracellular glycation occurs after the generation of the reactive 1,2-dicarbonyls methylglyoxal and glyoxal, and disruption of mitochondrial function is associated with hyperglycemia. However, the contribution of these reactive dicarbonyls to mitochondrial damage in pathology is unclear owing to uncertainties about their levels within mitochondria in cells and in vivo. To address this we have developed a mitochondria-targeted reagent (MitoG) designed to assess the levels of mitochondrial dicarbonyls within cells. MitoG comprises a lipophilic triphenylphosphonium cationic function, which directs the molecules to mitochondria within cells, and an o-phenylenediamine moiety that reacts with dicarbonyls to give distinctive and stable products. The extent of accumulation of these diagnostic heterocyclic products can be readily and sensitively quantified by liquid chromatography–tandem mass spectrometry, enabling changes to be determined. Using the MitoG-based analysis we assessed the formation of methylglyoxal and glyoxal in response to hyperglycemia in cells in culture and in the Akita mouse model of diabetes in vivo. These findings indicated that the levels of methylglyoxal and glyoxal within mitochondria increase during hyperglycemia both in cells and in vivo, suggesting that they can contribute to the pathological mitochondrial dysfunction that occurs in diabetes and aging.

β-Triketone inhibitors of plant p-hydroxyphenylpyruvate dioxygenase: modeling and comparative molecular field analysis of their interactions.

p-Hydroxyphenylpyruvate dioxygenase (HPPD) is the target site of beta-triketone herbicides in current use. Nineteen beta-triketones and analogues, including the naturally occurring leptospermone and grandiflorone, were synthesized and tested as inhibitors of purified Arabidopsis thaliana HPPD. The most active compound was a beta-triketone with a C(9) alkyl side chain, not reported as natural, which inhibited HPPD with an I(50) of 19 +/- 1 nM. This is significantly more active than sulcotrione, which had an I(50) of 250 +/- 21 nM in this assay system. The most active naturally occurring beta-triketone was grandiflorone, which had an I(50) of 750 +/- 70 nM. This compound is of potential interest as a natural herbicide because it can be extracted with good yield and purity from some Leptospermum shrubs. Analogues without the 1,3-diketone group needed to interact with Fe(2+) at the HPPD active site were inactive (I(50)s > 50 microM), as were analogues with prenyl or ethyl groups on the triketone ring. Modeling of the binding of the triketones to HPPD, three-dimensional QSAR analysis using CoMFA (comparative molecular field analysis), and evaluation of the hydrophobic contribution with HINT (hydropathic interactions) provided a structural basis to describe the ligand/receptor interactions.