Investigating the Contribution of Major Drug-Metabolising Enzymes to Possum-Specific Fertility Control.

The potential to improve the effectiveness and efficiency of potential oestrogen-based oral contraceptives (fertility control) for possums was investigated by comparing the inhibitory potential of hepatic CYP3A and UGT2B catalytic activity using a selected compound library (CYP450 inhibitor-based compounds) in possums to that of three other species (mouse, avian, and human). The results showed higher CYP3A protein levels in possum liver microsomes compared to other test species (up to a 4-fold difference). Moreover, possum liver microsomes had significantly higher basal p-nitrophenol glucuronidation activity than other test species (up to an 8-fold difference). However, no CYP450 inhibitor-based compounds significantly decreased the catalytic activity of possum CYP3A and UGT2B below the estimated IC50 and 2-fold IC50 values and were therefore not considered to be potent inhibitors of these enzymes. However, compounds such as isosilybin (65%), ketoconazole (72%), and fluconazole (74%) showed reduced UGT2B glucuronidation activity in possums, mainly at 2-fold IC50 values compared to the control (p < 0.05). Given the structural features of these compounds, these results could provide opportunities for future compound screening. More importantly, however, this study provided preliminary evidence that the basal activity and protein content of two major drug-metabolising enzymes differ in possums compared to other test species, suggesting that this could be further exploited to reach the ultimate goal: a potential target-specific fertility control for possums in New Zealand.

Key considerations when using the sulforhodamine B assay for screening novel anticancer agents.

Anticancer drug discovery programmes use a large number of in-vitro assays to screen the potency of compound libraries. The accuracy and reliability of these in-vitro assays are vital in selecting potent lead candidates for further (pre)clinical studies. Among the commonly used cell viability assays, the sulforhodamine B (SRB) assay has been a popular choice due to its simplicity, accuracy, reliability and reproducibility. SRB dye interacts with protein's basic amino acids and viable cell number is determined based on the cellular protein content. In this study, the cytotoxic potency of the novel hydroxythiopyridone derivatives towards A549 and H522 cells was determined using the SRB assay. The known drugs oxaliplatin and vorinostat were also examined. The resulting EC50 values were accurate, reliable and reproducible. However, all EC50 values calculated in 6-well plates were higher compared to those determined from 96-well plates. Furthermore, results from 6-well plates were also more variable compared to 96-well plates. Our results confirm that SRB assay is a reliable technique in screening the potency of anticancer drug candidates but plating conditions need to be carefully considered.

Does Crizotinib Auto-Inhibit CYP3A in vivo?

Crizotinib is a tyrosine kinase inhibitor used to treat anaplastic lymphoma kinase-positive lung cancer. There is in vitro evidence that crizotinib may auto-inhibit cytochrome P450 3A (CYP3A) activity, with important implications for crizotinib pharmacokinetics. In order to test whether crizotinib treatment alters CYP3A activity in vivo, mice were treated with 5 and 25 mg/kg crizotinib (p.o.) daily for 14 days. Results showed that crizotinib treatment did not alter CYP3A activity as determined by erythromycin N-demethylation. In addition, CYP3A polypeptide expression as measured by Western blot was unchanged. Therefore, our results do not support CYP3A inhibition by crizotinib in vivo.

A Multitargeted Approach: Organorhodium Anticancer Agent Based on Vorinostat as a Potent Histone Deacetylase Inhibitor.

The combination of more than one bioactive moiety in a multitargeted anticancer agent may result in synergistic activity of its components. Using this concept, bioorganometallic compounds were designed to feature a metal center, a 2-pyridinecarbothioamide (PCA), and a hydroxamic acid, which is found in the anticancer drug vorinostat (SAHA). The organometallics showed inhibitory activity in the nanomolar range against histone deacetylases (HDACs) as the key target for SAHA. In particular, the Rh complex was a potent inhibitor of HDAC6 over HDAC1 and HDAC8. Whereas this complex was highly cytotoxic in human cancer cells, it showed low toxicity in hemolysis studies and zebrafish, demonstrating the role of the metal center. For this complex a slightly reduced expression of vascular endothelial growth factor receptor 2 (VEGFR2) was established, which was upregulated by SAHA. This finding indicates that the new organometallics display different modes of action than their bioactive components.

Mechanisms of apoptosis modulation by curcumin: Implications for cancer therapy.

Cancer incidences are growing and cause millions of deaths worldwide. Cancer therapy is one of the most important challenges in medicine. Improving therapeutic outcomes from cancer therapy is necessary for increasing patients' survival and quality of life. Adjuvant therapy using various types of antibodies or immunomodulatory agents has suggested modulating tumor response. Resistance to apoptosis is the main reason for radioresistance and chemoresistance of most of the cancers, and also one of the pivotal targets for improving cancer therapy is the modulation of apoptosis signaling pathways. Apoptosis can be induced by intrinsic or extrinsic pathways via stimulation of several targets, such as membrane receptors of tumor necrosis factor-α and transforming growth factor-ß, and also mitochondria. Curcumin is a naturally derived agent that induces apoptosis in a variety of different tumor cell lines. Curcumin also activates redox reactions within cells inducing reactive oxygen species (ROS) production that leads to the upregulation of apoptosis receptors on the tumor cell membrane. Curcumin can also upregulate the expression and activity of p53 that inhibits tumor cell proliferation and increases apoptosis. Furthermore, curcumin has a potent inhibitory effect on the activity of NF-κB and COX-2, which are involved in the overexpression of antiapoptosis genes such as Bcl-2. It can also attenuate the regulation of antiapoptosis PI3K signaling and increase the expression of MAPKs to induce endogenous production of ROS. In this paper, we aimed to review the molecular mechanisms of curcumin-induced apoptosis in cancer cells. This action of curcumin could be applicable for use as an adjuvant in combination with other modalities of cancer therapy including radiotherapy and chemotherapy.

The aryl hydrocarbon receptor as an antitumor target of synthetic curcuminoids in colorectal cancer.

BACKGROUND: Curcumin has proven to be a potent antitumor agent in both preclinical and clinical models of colorectal cancer (CRC). It has also been identified as a ligand of the transcription factor known as the aryl hydrocarbon receptor (AHR). Our laboratory has identified the AHR as a mechanism which contributes to both tumorigenesis in a mouse model of inflammatory CRC as well an apoptotic target in vitro. Curcumin's role as an AHR ligand may modulate its effects to induce colon cancer cell death, and this role may be enhanced via structural modification of the curcumin backbone. We sought to determine if the two piperidone analogs of curcumin, RL66 and RL118, exhibit more robust antitumor actions than their parent compound in the context of colorectal cancer in vitro. Moreover, to ascertain the ability of curcumin, RL66 and RL118 to activate the AHR and evaluate if this activation has any effect on CRC cell death. MATERIALS AND METHODS: DLD1, HCT116, LS513, and RKO colon cell lines were propagated in vitro. Natural curcumin was obtained commercially, whereas RL66 and RL118 were synthesized and characterized de novo. Multiwell fluorescent/luminescent signal detection was used to simultaneously ascertain cell viability, cell cytonecrosis, and relative amounts of apoptotic activity. AHR activity was measured with a dual luciferase reporter gene system. Stable expression of small interfering RNA interference was established in the HCT116 cell lines to create AHR "knock down" cell lines. RESULTS: Both RL66 and RL118 proved to be more potent antitumor agents than their parent compound curcumin in all cell lines tested. The majority of this cell death was due to induction of apoptosis, which occurred earlier and to a greater degree following RL66 and RL118 treatment as opposed to curcumin. Also, RL66 and RL118 were found to be activators of AHR, and a portion of their ability to cause cell death was dependent on this induction. Curcumin was found unable to activate the AHR, and levels of AHR messenger RNA did not change their effects on cell death. CONCLUSIONS: Piperidone analogs of curcumin exhibited enhanced antitumor effects in vitro as opposed to their parent compound. Even more, this enhanced cell death profile may be partially attributed to the ability of these compounds to activate the AHR. Further study of synthetic curcumin analogs as chemopreventives and chemoadjuncts in CRC is warranted. Also, more generally, the AHR may represent a potential putative target for novel anticancer agents for CRC.

Utility of the second-generation curcumin analogue RL71 in canine histiocytic sarcoma.

Canine histiocytic sarcoma is an aggressive cancer, with a high rate of metastasis. Thus, novel therapeutic approaches are needed. Synthetic analogues of curcumin have elicited potent anti-cancer activity in multiple in vitro and in vivo models of human cancer. Furthermore, the compound 3,5-bis(3,4,5-trimethoxybenzylidene)-1-methylpiperidine-4-one (RL71) has recently exhibited potent cell cycle arrest and apoptotic induction in a canine osteosarcoma cell line. To determine its potency in canine histiocytic sarcoma cells, cell viability of DH82 and Nike cells was measured using the sulforhodamine B assay. Flow cytometry was then used to analyse both cell cycle distribution and apoptosis. Of the five curcumin analogues examined, RL71, had the highest potency with EC50 values of 0.66 ± 0.057 µM and 0.79 ± 0.13 µM in the DH82 and Nike cell lines, respectively. Furthermore, RL71 at the 1x EC50 concentration increased G2/M cell cycle arrest 2-fold, and at the 2x EC50 concentration increased the number of apoptotic cells 4-fold. These findings are consistent with previous work using RL71 in both canine and human cancer cell lines. Future research should be directed on time-dependent changes, and mechanistic investigation in greater detail to elucidate RL71 mechanisms of action.

The second-generation curcumin analogue RL71 elicits G2/M cell cycle arrest and apoptosis in canine osteosarcoma cells.

Canine osteosarcoma is an aggressive cancer, comprising 85% of canine bone neoplasms. Current treatment practices of surgery and chemotherapy increase 1-year survival by only 45%. The curcumin analogue RL71, has demonstrated potent in vitro and in vivo efficacy in several models of human breast cancer through increased apoptosis and cell cycle arrest. Thus, the present study aimed to investigate efficacy of curcumin analogues in two canine osteosarcoma cell lines. Osteosarcoma cell viability was assessed using the sulforhodamine B assay and mechanisms of action were determined by analysing the levels of cell cycle and apoptotic regulatory proteins via Western blotting. Further evidence was obtained using flow cytometry to detect cell cycle distribution and the number of apoptotic cells. RL71 was the most potent curcumin analogue with EC50 values of 0.64 ± 0.04 and 0.38 ± 0.009 µM (n = 3) in D-17 (commercial) and Gracie canine osteosarcoma cells, respectively. RL71 significantly increased the ratio of cleaved-caspase 3 to pro-caspase 3 and the level of apoptotic cells at the 2× and 5× EC50 concentration (p < 0.001, n = 3). Furthermore, at the same concentration, RL71 significantly increased the number of cells in the G2/M phase. In conclusion, RL71 has potent cytotoxic activity in canine osteosarcoma cells triggering G2/M arrest and apoptosis at concentrations achievable in vivo. Future research should further investigate molecular mechanisms for these changes in other canine osteosarcoma cell lines prior to in vivo investigation.

Characterisation of AMB-FUBINACA metabolism and CB1-mediated activity of its acid metabolite.

PURPOSE: AMB-FUBINACA is a synthetic cannabinoid receptor agonist (SCRA) which is primarily metabolised by hepatic enzymes producing AMB-FUBINACA carboxylic acid. The metabolising enzymes associated with this biotransformation remain unknown. This study aimed to determine if AMB-FUBINACA metabolism could be reduced in the presence of carboxylesterase (CES) inhibitors and recreational drugs commonly consumed with it. The affinity and activity of the AMB-FUBINACA acid metabolite at the cannabinoid type-1 receptor (CB1) was investigated to determine the activity of the metabolite. METHODS: The effect of CES1 and CES2 inhibitors, and delta-9-tetrahydrocannabinol (Δ9-THC) on AMB-FUBINACA metabolism were determined using both human liver microsomes (HLM) and recombinant carboxylesterases. Radioligand binding and cAMP assays comparing AMB-FUBINACA and AMB-FUBINACA acid were carried out in HEK293 cells expressing human CB1. RESULTS: AMB-FUBINACA was rapidly metabolised by HLM in the presence and absence of NADPH. Additionally, CES1 and CES2 inhibitors both significantly reduced AMB-FUBINACA metabolism. Furthermore, digitonin (100 µM) significantly inhibited CES1-mediated metabolism of AMB-FUBINACA by ~ 56%, while the effects elicited by Δ9-THC were not statistically significant. AMB-FUBINACA acid produced only 26% radioligand displacement consistent with low affinity binding. In cAMP assays, the potency of AMB-FUBINACA was ~ 3000-fold greater at CB1 as compared to the acid metabolite. CONCLUSIONS: CES1A1 was identified as the main hepatic enzyme responsible for the metabolism of AMB-FUBINACA to its less potent carboxylic acid metabolite. This biotransformation was significantly inhibited by digitonin. Since other xenobiotics may also inhibit similar SCRA metabolic pathways, understanding these interactions may elucidate why some users experience high levels of harm following SCRA use.

The piperazine analogue para-fluorophenylpiperazine alters timing of the physiological effects of the synthetic cannabinoid receptor agonist AMB-FUBINACA, without changing its discriminative stimulus, signalling effects, or metabolism.

AMB-FUBINACA is a synthetic cannabinoid receptor agonist (SCRA), which has been associated with substantial abuse and health harm since 2016 in many countries including New Zealand. A characteristic of AMB-FUBINACA use in New Zealand has included the observation that forensic samples (from autopsies) and drugs seized by police have often been found to contain para-fluorophenylpiperazine (pFPP), a relatively little-characterised piperazine analogue that has been suggested to act through 5HT1a serotonin receptors. In the current study, we aimed to characterise the interactions of these two agents in rat physiological endpoints using plethysmography and telemetry, and to examine whether pFPP altered the subjective effects of AMB-FUBINACA in mice trained to differentiate a cannabinoid (THC) from vehicle. Though pFPP did not alter the ability of AMB-FUBINACA to substitute for THC, it did appear to abate some of the physiological effects of AMB-FUBINACA in rats by delaying the onset of AMB-FUBINACA-mediated hypothermia and shortening duration of bradycardia. In HEK cells stably expressing the CB1 cannabinoid receptor, 5HT1a, or both CB1 and 5HT1a, cAMP signalling was recorded using a BRET biosensor (CAMYEL) to assess possible direct receptor interactions. Although low potency pFPP agonism at 5HT1a was confirmed, little evidence for signalling interactions was detected in these assays: additive or synergistic effects on potency or efficacy were not detected between pFPP and AMB-FUBINACA-mediated cAMP inhibition. Experiments utilising higher potency, classical 5HT1a ligands (agonist 8OH-DPAT and antagonist WAY100635) also failed to reveal evidence for mutual CB1/5HT1a interactions or cross-antagonism. Finally, the ability of pFPP to alter the metabolism of AMB-FUBINACA in rat and human liver microsomes into its primary carboxylic acid metabolite via carboxylesterase-1 was assessed by HPLC; no inhibition was detected. Overall, the effects we have observed do not suggest that increased harm/toxicity would result from the combination of pFPP and AMB-FUBINACA.

Identification of cyclohexanone derivatives that act as catalytic inhibitors of topoisomerase I: effects on tamoxifen-resistant MCF-7 cancer cells.

Breast cancer is commonly treated with anti-estrogens or aromatase inhibitors, but resistant disease eventually develops and new therapies for such resistance are of great interest. We have previously isolated several tamoxifen-resistant variant sub-lines of the MCF-7 breast cancer cell line and provided evidence that they arose from expansion of pre-existing minor populations. We have searched for therapeutic agents that exhibit selective growth inhibition of the resistant lines and here investigate 2,6-bis(pyridin-3-ylmethylene)-cyclohexanone (RL90) and 2,6-bis(pyridin-4-ylmethylene)-cyclohexanone (RL91). We found that two of the tamoxifen-resistant sub-lines (TamR3 and TamC3) unexpectedly showed increased sensitivity to RL90 and RL91. We utilized growth inhibition assays, flow cytometry and immunoblotting to establish a mechanistic basis for their action. Treated sensitive cells showed S-phase selective DNA damage, as detected by histone H2AX phosphorylation. Cellular responses were similar to those induced by the topoisomerase I poison camptothecin. Although IC(50) values of camptothecin, RL90, RL91 were correlated, studies with purified mammalian topoisomerase I suggested that RL90 and RL91 differed from camptothecin by acting as catalytic topoisomerase I inhibitors. These drugs provide a platform for the further development of DNA damaging drugs that have selective effects on tamoxifen resistant breast cancer cells. The results also raise the question of whether clinical topoisomerase I poisons such as irinotecan and topotecan might be active in the treatment of some types of tamoxifen-resistant cancer.

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

BACKGROUND: Curcumin 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. METHODS: The 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. RESULTS: The 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. CONCLUSIONS: These 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.

Raloxifene potentiates the effect of gefitinib in triple-negative breast cancer cell lines.

Triple-negative breast cancers (TNBCs) are characterized by a lack of approved targeted therapies and remain a challenge in the clinic. Several overexpressed proteins, including epidermal growth factor receptor (EGFR), have been associated with TNBCs and are considered potential therapeutic targets. However, EGFR inhibitors alone failed to demonstrate a cutting-edge advantage for treating TNBCs over conventional chemotherapies. Studies have shown that selective estrogen receptor modulators (SERMs) tamoxifen and raloxifene also affect TNBC cell viability. The combination of gefitinib and raloxifene was assessed against TNBC cell lines in vitro. Two TNBC cell lines, MDA-MB-231 and MDA-MB-468, were used to investigate the combination of gefitinib and raloxifene on cell viability, DNA synthesis, and apoptosis. The combination was assessed on intracellular signaling pathways, colony formation, migration, and angiogenesis. In the present study, raloxifene, in combination with gefitinib, decreased cell viability. The combination potentiates apoptosis and affects the expression and phosphorylation pattern of proteins involved in cell proliferation, such as NFκB, ß-catenin, and EGFR. Furthermore, evidence of apoptosis activation was also observed, along with a decreased cell migration and tumorigenicity of TNBC cells. Moreover, the combined treatment decreased the ability of neovascularization as assessed by tube formation of endothelial cells. These results suggested the potential of the combination of raloxifene and gefitinib for the prevention of TNBC growth and the appearance of metastatic events. Our findings provide the basis for future studies on the mechanism involved in raloxifene-gefitinib inhibition of ER-negative tumor growth.

Six fruit and vegetable peel beads for the simultaneous removal of heavy metals by biosorption.

In this study, a comparison between the biosorption performance of six fruit and vegetable peels, namely kiwifruit (KP), apple, banana, cucumber, orange and potato immobilized on sodium alginate beads has been made. Inductively coupled plasma coupled with mass spectroscopy was used for measuring the concentration of metal ions in solution before and after biosorption. A range of kinetic models were also applied to the biosorption batch data. The results showed that biosorption percentage of the ions were different on the various beads. For example, the decreasing order of biosorption by one KP bead at equilibrium was Cd > Cu > Hg > Ni > Pb > Cr > As, with approximately 92%, 84%, 80%, 75%, 67%, 34%, and 17% simultaneous removal of ions, respectively. The fastest biosorption was seen with Cd and Pb, as both reached equilibrium by 24 h. Equilibrium time of all other ions occurred by 48 h. While all beads in their unmodified form were suitable for the removal of divalent cations, KP bead showed significantly higher removal of the anion hexavalent Cr. Biosorption of Cd, Hg and Ni was limited by both pseudo-first order and pseudo-second order reaction rates. For Cr and Cu, the reaction was controlled by film diffusion and pseudo-first order rates. At a higher solution concentration, the preference of ions biosorbed as well as their percentage removed changed. Overall, the results indicated that KP beads show promise as a cost-effective method for removing toxic ions by biosorption, especially hexavalent chromium from drinking water.

Exploring the Mechanism and Suggesting Combination Therapies for HDAC Inhibitors in Androgen Receptor-Null Prostate Cancer Using Multivariate Statistical Analysis and Data Mining Techniques.

Previously, we showed that novel histone deacetylase (HDAC) inhibitors, N1-hydroxy-N 8-(4-(pyridine-2-carbothioamido)phenyl)octanediamide (Jazz90) and [chlorido(η5-pentamethylcyclopentadienyl)(N1-hydroxy-N8-(4-(pyridine-2-carbothioamido-κ2 N, S)phenyl)octanediamide)rhodium(III)] chloride (Jazz167), have cytostatic and anti-angiogenic effects in androgen receptor-negative prostate cancer cells and are also non-toxic in BALB/c mice. However, only univariate statistical analysis was carried out to determine the role of individual proteins. In this study, multivariate statistical analyses (MVAs) and data mining procedures were carried out with the objective of determining the molecular networks that explain the growth inhibitory potential of Jazz90 and Jazz167 in PC3 cells and to determine potential inhibitors that can be used in combination with these HDAC inhibitors. Lasso regression revealed that angiogenic factors, vascular endothelial growth factor-A (VEGF-A), and vascular endothelial growth factor receptor-2 (VEGFR-2), alongside HDAC inhibition, predicted the reduction in cell number with an adjusted R 2 value of 0.99 following Jazz90 treatment, whereas VEGFR-2, acetylation of histone-3, and HDAC inhibition predicted cell number with an adjusted R 2 value of 0.84 following Jazz167 treatment. These results were further followed up with ridge regression, hierarchical cluster analysis, random forest classification (RFC), and support vector machines. RFC and support vector machines also predicted the treatment groups with a 100% accuracy. MVAs also revealed that Jazz90 should be examined in combination with epithelial to mesenchymal transitioning inhibitors, such as simvastatin and olaparib, whereas Jazz167 should be examined with venetoclax or navitoclax. Future studies should also address the roles of VEGF-A and VEGFR-2 in cellular proliferation, whereas p27 function should be examined for its role in PC3 cell migration.

Characterization of marine-derived halogenated indoles as ligands of the aryl hydrocarbon receptor.

The aryl hydrocarbon receptor (AhR), a ligand-activated transcription factor thought to mediate a number of physiological roles in the body, is becoming a target of interest for the development of new therapeutics. However, previous research has demonstrated that the downstream effects of AhR ligands cannot be predicted based simply on whether a ligand acts as an agonist or antagonist and the persistence of AhR signaling is thought to be a key determining feature. The current study investigated the AhR activity of four halogenated indoles isolated from the New Zealand red alga, Rhodophyllis membranacea: 4,7-dibromo-2,3-dichloroindole (4DBDCI), 7-bromo-2,3-dichloro-6-iodoindole (BDCII), 6,7-dibromo-2,3-dichloroindole (6DBDCI) and 2,6,7-tribromo-3-chloroindole (TBCI). Their ability to activate AhR signaling, measured as CYP1A1 activity via the ethoxyresorufin O-deethylase (EROD) assay, was determined in human HepG2, mouse Hepa1c1c7 and rat H4IIE liver cancer cells. All four compounds induced CYP1A1 activity in HepG2 cells, suggesting they all acted as AhR agonizts. 4DBDCI was particularly efficacious, inducing an 11-fold increase. Hepa1c1c7 and H4IIE cells, however, were generally less responsive to the halogenated indoles. All four compounds were persistent AhR agonizts, inducing peak CYP1A1 activity after 72 h. Moreover, the 2,3,6,7-substituted BDCII, 6DBDCI and TBCI, but not 4DBDCI, competed with 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) for AhR binding as observed by the inhibition of TCDD-induced CYP1A1 activity. Overall, the current study has characterized four previously untested AhR ligands, highlighting differences in species sensitivity and persistence of signaling to provide a framework for their potential future use.

Raloxifene Suppresses Tumor Growth and Metastasis in an Orthotopic Model of Castration-Resistant Prostate Cancer.

Androgen receptor (AR)-castrate-resistant prostate cancer (CRPC) is an aggressive form of prostate cancer that does not have clinically approved targeted treatment options. To this end, the cytotoxic potential of raloxifene and the synthetic curcumin derivative 2,6-bis (pyridin-4-ylmethylene)-cyclohexanone (RL91) was examined in AR-(PC3 and DU145) cells and AR+ (LnCaP) CRPC cells. The results showed that both raloxifene and RL91 elicited significant cytotoxicity across three cell lines with the lowest EC50 values in PC3 cells. Additionally, the two drugs were synergistically cytotoxic toward the PC3, DU-145 and LNCaP cell lines. To determine the effect of the drug combination in vivo, an orthotopic model of CRPC was used. Male mice were injected with PC3 prostate cancer cells and then treated with vehicle (5 mL/kg), raloxifene (8.5 mg/kg, po), RL91 (8.5 mg/kg, po) or a combination of raloxifene and RL91 for six weeks. Sham animals were subjected to the surgical procedure but were not implanted with PC3 cells. The results showed that raloxifene decreased tumor size and weight as well as metastasis to renal lymph nodes. However, combination treatment reversed the efficacy of raloxifene as tumor volume and metastasis returned to control levels. The results suggest that raloxifene has tumor suppressive and anti-metastatic effects and has potential for further clinical use in AR-CRPC.

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

Estrogen 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 EC(50) values of 1.54 and 1.10 µM, respectively, in MDA-MB-231 cells and EC(50) values of 0.51 and 0.23 in SKBr3 cells. All other new compounds examined were less potent than curcumin, which elicited EC(50) values of 7.6 and 2.4 µM in MDA-MB-231 and SKBr3 cells, respectively. Mechanistic analyses demonstrated that RL90 and RL91 significantly induced G(2)/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-3ß transiently decreased, while ß-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.

Cucumber peel bead biosorbent for multi-ion decontamination of drinking water collected from a mine region in New Zealand.

Cucumber peel as a bead was examined for its ability to remove heavy metals from drinking water. Deionised laboratory water was spiked with seven toxic ions namely, arsenic, cadmium, chromium, copper, mercury, lead and nickel at 0.1 mg L-1 and kinetic studies were performed over 72 h. Kinetic data were modelled using film diffusion, pore diffusion, Weber-Morris, pseudo-first-order, pseudo-second-order and Elovich equation. The bead surface was imaged before and after biosorption using scanning electron microscopy coupled with energy dispersive spectroscopy (EDS). Results indicated that different ions contained in a multi-ion solution were biosorbed by different mechanisms and at different rates. Equilibrium biosorption for Cd, Hg and Ni was ∼91, 90 and 67%, respectively, at 24 h. These ions diffused through the pores of the bead, as they were not identified by EDS, and their biosorption increased with an increase in temperature. The least biosorbed ions were As and Cr with ∼21 and 17% equilibrium biosorption, respectively. The removal of only Cu, Hg, Pb and Ni was pH-dependent. Cucumber peel beads removed all spiked ions from real drinking water collected near the Macraes gold mine in New Zealand, but the biosorption percentage was lower for Cd, Cu, Pb and Ni compared to spiked deionised laboratory water. The results of this study suggest that cucumber peel when immobilised on a sodium alginate bead can be used as a potential biosorbent for the removal of multiple toxic ions from drinking water and their use warrants further examination in contaminated drinking water.

In Vitro Hepatic Assessment of Cineole and Its Derivatives in Common Brushtail Possums (Trichosurus vulpecula) and Rodents.

Folivore marsupials, such as brushtail possum (Trichosurus Vulpecula) and koala (Phascolarctos cinereus), can metabolise higher levels of dietary terpenes, such as cineole, that are toxic to eutherian mammals. While the highly efficient drug metabolising enzymes, cytochrome P450 3A (CYP3A) and phase II conjugating enzymes (UDP-glucuronosyltransferase, UGT), are involved in the metabolism of high levels of dietary terpenes, evidence for inhibitory actions on these enzymes by these terpenes is scant. Thus, this study investigated the effect of cineole and its derivatives on catalytic activities of hepatic CYP3A and UGT in mice, rats, and possums. Results showed that cineole (up to 50 µM) and its derivatives (up to 25 µM) did not significantly inhibit CYP3A and UGT activities in mice, rats, and possums (both in silico and in vitro). Interestingly, basal hepatic CYP3A catalytic activity in the possums was ~20% lower than that in rats and mice. In contrast, possums had ~2-fold higher UGT catalytic activity when compared to mice and rats. Thus, these basal enzymatic differences may be further exploited in future pest management strategies.