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Oxidative damage and accumulation of extracellular matrix (ECM) components play a crucial role in the adverse outcome of cardiac hypertrophy. Evidence suggests that nuclear factor erythroid-derived factor 2 related factor 2 (Nrf2) can modulate oxidative damage and adverse myocardial remodeling. Daphnetin (Daph) is a coumarin obtained from the plant genus Daphne species that exerts anti-oxidative and anti-inflammatory properties. Herein, we investigated the roles of Daph in transverse aortic constriction (TAC)-induced cardiac hypertrophy and fibrosis in mice. TAC-induced alterations in cardiac hypertrophy markers, histopathological changes, and cardiac function were markedly ameliorated by oral administration of Daph in mice. We found that Daph significantly reduced the reactive oxygen species (ROS) generation, increased the nuclear translocation of Nrf2, and consequently, reinstated the protein levels of NAD(P)H quinone dehydrogenase1 (NQO1), heme oxygenase-1 (HO-1), and other antioxidants in the heart. Besides, Daph significantly inhibited the TAC-induced accumulation of ECM components, including α-smooth muscle actin (α-SMA), collagen I, collagen III, and fibronectin, and interfered with the TGF-β1/Smad2/3 signaling axis. Further studies revealed that TAC-induced terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) positive nuclei and the protein levels of Bax/Bcl2 ratio and cleaved caspase 3 were substantially decreased by Daph treatment. We further characterized the effect of Daph on angiotensin II (Ang-II)-stimulated H9c2 cardiomyoblast cells and observed that Daph markedly decreased the Ang-II induced increase in cell size, production of ROS, and proteins associated with apoptosis and fibrosis. Mechanistically, Daph alone treatment enhanced the protein levels of Nrf2, NQO1, and HO-1 in H9c2 cells. The inhibition of this axis by Si-Nrf2 transfection abolished the protective effect of Daph in H9c2 cells. Taken together, Daph effectively counteracted the TAC-induced cardiac hypertrophy and fibrosis by improving the Nrf2/HO-1 axis and inhibiting the TGF-β1/Smad2/3 signaling axis.

Treatment of melanoma with a BRAF inhibitor (BRAFi) frequently initiates development of BRAFi resistance, leading to increased tumor progression and metastasis. Previously, we showed that combined inhibition of elevated WNT5A and IL-6 signaling reduced the invasion and migration of BRAFi-resistant (BRAFi-R) melanoma cells. However, the use of a combined approach per se and the need for high inhibitor concentrations to achieve this effect indicate a need for an alternative and single target. One such target could be myristoylated alanine-rich C-kinase substrate (MARCKS), a downstream target of WNT5A in BRAFi-sensitive melanoma cells. Our results revealed that MARCKS protein expression and activity are significantly elevated in PLX4032 and PLX4720 BRAFi-R A375 and HTB63 melanoma cells. Surprisingly, neither WNT5A nor IL-6 contributed to the increases in MARCKS expression and activity in BRAFi-R melanoma cells, unlike in BRAFi-sensitive melanoma cells. However, despite the above findings, our functional validation experiments revealed that MARCKS is essential for the increased metastatic behavior of BRAFi-R melanoma cells. Knockdown of MARCKS in BRAFi-R melanoma cells caused reductions in the F-actin content and the number of filopodia-like protrusions, explaining the impaired migration, invasion and metastasis of these cells observed in vitro and in an in vivo zebrafish model. In our search for an alternative explanation for the increased activity of MARCKS in BRAFi-R melanoma cells, we found elevated basal activities of PKCα, PKCε, PKCι, and RhoA. Interestingly, combined inhibition of basal PKC and RhoA effectively impaired MARCKS activity in BRAFi-R melanoma cells. Our results reveal that MARCKS is an attractive single antimetastatic target in BRAFi-R melanoma cells.

Treatment of melanoma with a BRAF inhibitor (BRAFi) frequently initiates development of BRAFi resistance, leading to increased tumor progression and metastasis. Previously, we showed that combined inhibition of elevated WNT5A and IL-6 signaling reduced the invasion and migration of BRAFi-resistant (BRAFi-R) melanoma cells. However, the use of a combined approach per se and the need for high inhibitor concentrations to achieve this effect indicate a need for an alternative and single target. One such target could be myristoylated alanine-rich C-kinase substrate (MARCKS), a downstream target of WNT5A in BRAFi-sensitive melanoma cells. Our results revealed that MARCKS protein expression and activity are significantly elevated in PLX4032 and PLX4720 BRAFi-R A375 and HTB63 melanoma cells. Surprisingly, neither WNT5A nor IL-6 contributed to the increases in MARCKS expression and activity in BRAFi-R melanoma cells, unlike in BRAFi-sensitive melanoma cells. However, despite the above findings, our functional validation experiments revealed that MARCKS is essential for the increased metastatic behavior of BRAFi-R melanoma cells. Knockdown of MARCKS in BRAFi-R melanoma cells caused reductions in the F-actin content and the number of filopodia-like protrusions, explaining the impaired migration, invasion and metastasis of these cells observed in vitro and in an in vivo zebrafish model. In our search for an alternative explanation for the increased activity of MARCKS in BRAFi-R melanoma cells, we found elevated basal activities of PKCα, PKCε, PKCι, and RhoA. Interestingly, combined inhibition of basal PKC and RhoA effectively impaired MARCKS activity in BRAFi-R melanoma cells. Our results reveal that MARCKS is an attractive single antimetastatic target in BRAFi-R melanoma cells.

Among breast cancer subtypes, the triple negative breast cancer (TNBC) has the worst prognosis. In absence of any permitted targeted therapy, standard chemotherapy is the mainstay for TNBC treatment. Hence, there is a crucial need to identify potential druggable targets in TNBCs for its effective treatment. In recent times, metabolic reprogramming has emerged as cancer cells hallmark, wherein cancer cells display discrete metabolic phenotypes to fuel cell progression and metastasis. Altered glycolysis is one such phenotype, in which even in oxygen abundance majority of cancer cells harvest considerable amount of energy through elevated glycolytic-flux. In the present review, we attempt to summarize the role of key glycolytic enzymes i.e. HK, Hexokinase; PFK, Phosphofructokinase; PKM2, Pyruvate kinase isozyme type 2; and LDH, Lactate dehydrogenase in TNBCs, and possible therapeutic options presently available.

WNT5A is a well-known mediator of melanoma cell invasion and metastasis via its ability to activate protein kinase C (PKC), which is monitored by phosphorylation of the endogenous PKC substrate myristoylated alanine-rich c-kinase substrate (MARCKS). However, a possible direct contribution of MARCKS in WNT5A-mediated melanoma cell invasion has not been investigated. Analyses of melanoma patient databases suggested that similar to WNT5A expression, MARCKS expression appears to be associated with increased metastasis. A relationship between the two is suggested by the findings that recombinant WNT5A (rWNT5A) induces both increased expression and phosphorylation of MARCKS, whereas WNT5A silencing does the opposite. Moreover, WNT5A-induced invasion of melanoma cells was blocked by siRNA targeting MARCKS, indicating a crucial role of MARCKS expression and/or its phosphorylation. Next, we employed a peptide inhibitor of MARCKS phosphorylation that did not affect MARCKS expression and found that it abolished WNT5A-induced melanoma cell invasion. Similarly, rWNT5A induced the accumulation of phosphorylated MARCKS in membrane protrusions at the leading edge of melanoma cells. Our results demonstrate that WNT5A-induced phosphorylation of MARCKS is not only an indicator of PKC activity but also a crucial regulator of the metastatic behavior of melanoma and therefore an attractive future antimetastatic target in melanoma patients.

Increased expression and signalling of WNT5A and interleukin-6 (IL-6) have both been shown to promote melanoma progression. Here, we investigated the proposed existence of a WNT5A-IL-6 positive feedback loop that drives melanoma migration and invasion. First, the HOPP algorithm revealed that the invasive phenotype of cultured melanoma cells was significantly correlated with increased expression of WNT5A or IL-6. In three invasive melanoma cell lines, endogenous WNT5A protein expression was related to IL-6 protein secretion. Knockdown with anti-IL-6 siRNAs or treating WM852 melanoma cells with a neutralising anti-IL-6 antibody reduced WNT5A protein expression. Conversely, the silencing of WNT5A expression by WNT5A siRNAs or treating WM852 melanoma cells with Box5 (a WNT5A antagonist) significantly reduced IL-6 secretion. Interestingly, these effects occurred at the protein level but not at the transcriptional levels. Functionally, we demonstrated that combined siRNA knockdown of WNT5A and IL-6 expression or the simultaneous inhibition of WNT5A and IL-6 signalling inhibited melanoma cell invasion more effectively than suppressing each factor individually. Together, our results demonstrate that WNT5A and IL-6 are connected through a positive feedback loop in melanoma cells and that the combined targeting of both molecules could serve as an effective therapeutic means to reduce melanoma metastasis.

Despite the clinical development of novel adjuvant and neoadjuvant chemotherapeutic drugs, metastatic breast cancer is one of the leading causes of cancer-related death among women. The present review focuses on the relevance, mechanisms, and therapeutic potential of targeting WNT5A as a future anti-metastatic treatment strategy for breast cancer patients by restoring WNT5A signaling as an innovative therapeutic option. WNT5A is an auto- and paracrine β-catenin-independent ligand that has been shown to induce tumor suppression as well as oncogenic signaling, depending upon cancer type. In breast cancer patients, WNT5A protein expression has been observed to be significantly reduced in between 45 and 75% of the cases and associated with early relapse and reduced disease-free survival. WNT5A triggers various downstream signaling pathways in breast cancer that primarily affect tumor cell migration and invasion. The accumulated in vitro results reveal that treatment of WNT5A-negative breast cancer cells with recombinant WNT5A caused different tumor-suppressive responses and in particular it impaired migration and invasion. The anti-migratory/invasive and anti-metastatic effects of reconstituting WNT5A signaling by the small WNT5A mimicking peptide Foxy5 form the basis for two successful clinical phase 1-studies aiming at determining safety and pharmacokinetics as well as defining dose-level for a subsequent phase 2-study. We conclude that re-installation of WNT5A signaling is an attractive and promising anti-metastatic therapeutic approach for future treatment of WNT5A-negative breast cancer patients.

In recent years, scientists have advocated the use of targeted therapies in the form of drugs that modulate genes and proteins that are directly associated with cancer progression and metastasis. Malignant melanoma is a dreadful cancer type that has been associated with the rapid dissemination of primary tumors to multiple sites, including bone, brain, liver and lungs. The discovery that approximately 40%-50% of malignant melanomas contain a mutation in BRAF at codon 600 gave scientists a new approach to tackle this disease. However, clinical studies on patients have shown that although BRAFi (BRAF inhibitors) trigger early anti-tumor responses, the majority of patients later develop resistance to the therapy. Recent studies have shown that WNT5A plays a key role in enhancing the resistance of melanoma cells to BRAFi. The focus of the current review will be on melanoma development, signaling pathways important to acquired resistance to BRAFi, and why WNT5A inhibitors are attractive candidates to be included in combinatorial therapies for melanoma.

Combination therapy using two or more small molecule inhibitors of aberrant signaling cascade in aggressive breast cancers is a promising therapeutic strategy over traditional monotherapeutic approaches. Here, we have studied the synergistic mechanism of resveratrol and curcumin induced apoptosis using in vitro (cigarette smoke condensate mediated transformed breast epithelial cell, MCF-10A-Tr) and in vivo (tumor xenograft mice) model system. Resveratrol exposure increased the intracellular uptake of curcumin in a dose dependent manner and caused apoptosis in MCF-10A-Tr cells. Approximately, ten fold lower IC50 value was noted in cells treated with the combination of resveratrol (3μM) and curcumin (3μM) in comparison to 30μM of resveratrol or curcumin alone. Resveratrol+curcumin combination caused apoptosis by increasing Bax/Bcl-xL ratio, Cytochrome C release, cleaved product of PARP and caspase 3 in cells. Interestingly, this combination unaltered the protein expressions of WNT-TCF and Notch signaling components, β-catenin and cleaved notch-1 val1744, respectively. Furthermore, the combination also significantly decreased the intermediates of Hedgehog-Gli cascade including SMO, SHH, Gli-1, c-MYC, Cyclin-D1, etc. and increased the level of p21(Waf/Cip1) in vitro and in vivo. A significant reduction of Gli- promoter activity was noted in combinational drug treated cells in comparison to individual drug treatment. Un-alteration of the expressions of the above proteins and Gli1 promoter activity in p21(Waf/Cip1) knockout cells suggests this combination caused apoptosis through p21(Waf/Cip1). Thus, our findings revealed resveratrol and curcumin synergistically caused apoptosis in cigarette smoke induced breast cancer cells through p2(Waf/Cip1) mediated inhibition of Hedgehog-Gli cascade.

Based on the pharmacophoric features of the natural product combretastatin A-4 (CA-4) and its synthetic analogues that inhibit tubulin polymerization, a series of novel 2-aryl-3-arylamino-imidazo-pyridines/pyrazines as potential antitubulin anticancer agents were designed. They were synthesized by a one-pot method involving preparation of isocyanides from the anilines via formylation and subsequent dehydration followed by their reactions with heterocyclic-2-amidines and aldehydes. Compounds 1, 2, 14, and 15 were found to exhibit significant tubulin polymerization inhibition and disruption of tubulin–microtubule dynamics similar to that of CA-4. They showed potent anticancer activities in kidney, breast and cervical cancer cell lines, and relatively low toxicity to normal cells, compared to CA-4. The compounds induced DNA and chromosomal damage, and apoptosis via cell cycle arrest in the G2/M phase. The molecular docking and molecular dynamics (MD) simulation studies revealed that disruption of microtubule dynamics might occur by interaction of the compounds at the colchicine binding site of the α,β-tubulin heterodimer interface, similar to that of CA-4. Molecular modelling analysis showed that two of the three methoxy groups at ring A of all four potent compounds (1, 2, 14, and 15) were involved in bifurcated hydrogen bonding with Cysβ241, an important molecular recognition interaction to show tubulin inhibitory activity. In comparison to CA-4, the bridging NH and the imidazo-pyridine/pyrazine moieties in the title compounds provide flexibility for attaining the required dihedral relationship of two aryls and additional pharmacophoric features required for the interaction with the key residues of the colchicine binding site.

Cigarette smoking is a crucial factor in the development and progression of multiple cancers including breast. Here, we report that repeated exposure to a fixed, low dose of cigarette smoke condensate (CSC) prepared from Indian cigarettes is capable of transforming normal breast epithelial cells, MCF-10A, and delineate the biochemical basis for cellular transformation. CSC transformed cells (MCF-10A-Tr) were capable of anchorage-independent growth, and their anchorage dependent growth and colony forming ability were higher compared to the non-transformed MCF-10A cells. Increased expression of biomarkers representative of oncogenic transformation (NRP-1, Nectin-4), and anti-apoptotic markers (PI3K, AKT, NFκB) were also noted in the MCF-10A-Tr cells. Short tandem repeat (STR) profiling of MCF-10A and MCF-10A-Tr cells revealed that transformed cells acquired allelic variation during transformation, and had become genetically distinct. MCF-10A-Tr cells formed solid tumors when implanted into the mammary fat pads of Balb/c mice. Data revealed that CSC contained approximately 1.011μg Cd per cigarette equivalent, and Cd (0.0003μg Cd/1×10(7) cells) was also detected in the lysates from MCF-10A cells treated with 25μg/mL CSC. In similar manner to CSC, CdCl2 treatment in MCF-10A cells caused anchorage independent colony growth, higher expression of oncogenic proteins and increased PI3K-AKT-NFκB protein expression. An increase in the expression of PI3K-AKT-NFκB was also noted in the mice xenografts. Interestingly, it was noted that CSC and CdCl2 treatment in MCF-10A cells increased ROS. Collectively, results suggest that heavy metals present in cigarettes of Indian origin may substantially contribute to tumorigenesis by inducing intercellular ROS accumulation and increased expression of PI3K, AKT and NFκB proteins.

Aim: The authors have systematically investigated the anticancer potentiality of silver-based nanoparticles (AgNPs) and the mechanism underlying their biological activity in human colon cancer cells. Materials & methods: Starch-capped AgNPs were synthesized, characterized and their biological activity evaluated through multiple biochemical assays. Results: AgNPs decreased the growth and viability of HCT116 colon cancer cells. AgNP exposure increased apoptosis, as demonstrated by an increase in 4´,6-diamidino-2-phenylindole-stained apoptotic nuclei, BAX/BCL-XL ratio, cleaved poly(ADP-ribose) polymerase, p53, p21 and caspases 3, 8 and 9, and by a decrease in the levels of AKT and NF-κB. The cell population in the G1 phase decreased, and the S-phase population increased after AgNP treatment. AgNPs caused DNA damage and reduced the interaction between p53 and NF-κB. Interestingly, no significant alteration was noted in the levels of p21, BAX/BCL-XL and NF-κB after AgNP treatment in a p53-knockout HCT116 cell line. Conclusion: AgNPs are bona fide anticancer agents that act in a p53-dependent manner. Original submitted 16 March 2012; Revised submitted 25 August 2012; Published online 21 March 2013

Scaffold hybridization of several natural and synthetic anticancer leads led to the consideration of indenoindolones as potential novel anticancer agents. A series of these compounds were prepared by a diversity-feasible synthetic method. They were found to possess anticancer activities with higher potency compared to etoposide and 5-fluorouracil in kidney cancer cells (HEK 293) and low toxicity to corresponding normal cells (Vero). They exerted apoptotic effect with blocking of cell cycle at G2/M phase.

Pentacyclic Triterpenoids (PTs) and their analogues as well as derivatives are emerging as important drug leads for various diseases. They act through a variety of mechanisms and a majority of them inhibit the nuclear factor kappa-beta (NF-κB) signaling pathway. In this study, we examined the effects of the naturally occurring PTs on IκB kinase-β (IKKβ), which has great scientific relevance in the NF-κB signaling pathway. On virtual screening, 109 PTs were screened through the PASS (prediction of activity spectra of substances) software for prediction of NF-κB inhibitory activity followed by docking on the NEMO/IKKβ association complex (PDB: 3BRV) and testing for compliance with the softened Lipinski's Rule of Five using Schrodinger (LLC, New York, USA). Out of the projected 45 druggable PTs, Corosolic Acid (CA), Asiatic Acid (AA) and Ursolic Acid (UA) were assayed for IKKβ kinase activity in the cell free medium. The UA exhibited a potent IKKβ inhibitory effect on the hotspot kinase assay with IC50 of 69 μM. Whereas, CA at 50 μM concentration markedly reduced the NF-κB luciferase activity and phospho-IKKβ protein expressions. The PTs tested, attenuated the expression of the NF-κB cascade proteins in the LPS-stimulated RAW 264.7 cells, prevented the phosphorylation of the IKKα/β and blocked the activation of the Interferon-gamma (IFN-γ). The results suggest that the IKKβ inhibition is the major mechanism of the PTs-induced NF-κB inhibition. PASS predictions along with in-silico docking against the NEMO/IKKβ can be successfully applied in the selection of the prospective NF-κB inhibitory downregulators of IKKβ phosphorylation.

Colorectal cancer (CRC) patients with APC mutations do not benefit from 5-FU therapy. It was reported that APC physically interacts with POLβ and FEN1, thus blocking LP-BER via APC's DNA repair inhibitory (DRI) domain in vitro. The aim of this study was to elucidate how APC status affects BER and the response of CRC to 5-FU. HCT-116, HT-29, and LOVO cells varying in APC status were treated with 5-FU to evaluate expression, repair, and survival responses. HCT-116 expresses wild-type APC; HT-29 expresses an APC mutant that contains DRI domain; LOVO expresses an APC mutant lacking DRI domain. 5-FU increased the expression of APC and decreased the expression of FEN1 in HCT-116 and HT-29 cells, which were sensitized to 5-FU when compared to LOVO cells. Knockdown of APC in HCT-116 rendered cells resistant to 5-FU, and FEN1 levels remained unchanged. Re-expression of full-length APC in LOVO cells caused sensitivity to 5-FU, and decreased expression of FEN1. These knockdown and addback studies confirmed that the DRI domain is necessary for the APC-mediated reduction in LP-BER and 5-FU. Modelling studies showed that 5-FU can interact with the DRI domain of APC via hydrogen bonding and hydrophobic interactions. 5-FU resistance in CRC occurs with mutations in APC that disrupt or eliminate the DRI domain's interaction with LP-BER. Understanding the type of APC mutation should better predict 5-FU resistance in CRC than simply characterizing APC status as wild-type or mutant.

Here, we report the p53 dependent mitochondria- mediated apoptotic mechanism of plant derived silver- nanoparticle (PD-AgNPs) in colorectal cancer cells (CRCs). PD-AgNPs was synthesized by reduction of AgNO3 with leaf extract of a medicinal plant periwinkle and characterized. Uptake of PD-AgNPs (ξ - 2.52±4.31 mV) in HCT116 cells was 3 fold higher in comparison to synthetic AgNPs (ξ + 2.293±5.1 mV). A dose dependent increase in ROS produc- tion, activated JNK and decreased mitochondrial membrane potential (MMP) were noted in HCT116 but not in HCT116 p53 �/� cells after PD-AgNP exposure. PD-AgNP-mediated apoptosis in CRCs is a p53 dependent process involving ROS and JNK cascade.

Two different microbial biosurfactants S9BS and CHBS were isolated from Lysinibacillus fusiformis S9 and Bacillus tequilensis CH. Cytotoxicity effect of these biosurfactants on human embryonic kidney cancerous cell (HEK-293) were studied with the help of 3-(4,5-dimethylthiazol-2yl-)-2, 5-diphenyl tetrazolium bromide (MTT) assay and morphological changes were observed under inverted microscope. The biosurfactants exhibited positive cytotoxic effect on HEK-293 cell line. It was found that LC50 of S9BS and CHBS were 75 and 100 μg ml(-1), respectively. Further cell cycle and apoptosis analysis of biosurfactant-treated HEK-293 cell line were done by FACS. In this study, cytotoxic effect of glycolipid biosurfactant against HEK-293 cell lines is reported for the first time. Mechanism towards increased membrane permeability of biosurfactant-treated cancer cell may be the incorporation of its lipid moiety into the plasma membrane leading to formation of pores and membrane disruption. Hence, these microbial biosurfactants can prove to be significant biomolecule for cancer treatment.

We have previously reported that 4-(3-(tert-butylamino)imidazo[1,2-α]pyridine-2-yl)benzoic acid, a bicyclic N-fused aminoimidazoles derivative (BNFA-D), possesses anticancer potentiality against breast and kidney cancer cells with minimal toxicities to corresponding normal cells. Here, we explored the mechanism of action of BNFA-D in breast cancer cells using multiple cell-based assays such as MTT, DAPI, FACS, Western blot, and immunoprecipitation. BNFA-D caused apoptosis by upregulating PTEN leading to inhibition of Wnt/TCF signaling cascade and arresting S phase in breast cancer cells. Expression levels of β-catenin, cyclin D1, C-MYC, and phospho-AKT (Ser473) decreased with simultaneous increase in the levels of GSK3β, CK1, and PTEN in BNFA-D-treated MCF-7 cells. Interestingly, silencing of PTEN in breast cancer cells reversed the phenomenon of Wnt/TCF signaling cascade inhibition after BNFA-D treatment.

An approach involving rational structural elaboration of the biologically active natural product diindolylmethane (DIM) with the incorporation of aminophosphonate and urea moieties toward the discovery of potent anticancer agents was considered. A four-step approach for the synthesis of DIM aminophosphonate and urea derivatives was established. These novel compounds showed potent anticancer activities in two representative kidney and colon cancer cell lines, low toxicity to normal cells, higher potency than the parent natural product DIM and etoposide, and potent inhibition of cancer cell migration. Biophysical and immunological studies, including DAPI nuclear staining, western blot analysis with apoptotic protein markers, flow cytometry, immunocytochemistry, and comet assays of the two most potent compounds revealed good efficacies in apoptosis and DNA damage. It was found that down-regulation of nuclear factor κB (NF-κB p65) could be an important mode of action in apoptosis, and the two most potent derivatives were found to be more potent than parent compound DIM in the down-regulation of NF-κB. Our results show the importance of structural elaboration of DIM by rational incorporation of aminophosphonate and urea moieties to produce potent anticancer agents; they also suggest that this approach using other structurally simple bioactive natural products as scaffolds holds promise for future drug discovery and development.

We previously showed that quinacrine (QC), a small molecule antimalarial agent, also presented anticancer activity in breast cancer cells through activation of p53, p21, and inhibition of topoisomerase activity. Here we have systematically studied the detailed cell death mechanism of this drug using three colon cancer cell lines (HCT-116 parental, isogenic HCT-116 p53-/-, and HCT-116 p21-/- sublines). QC caused a dose-dependent reduction in cell viability in all three cell lines. However, the parental cells were more susceptible to QC-mediated cell death, suggesting that p53- and p21-dependent processes were involved. QC-mediated cell death was measured with the following endpoints: the Bax/Bcl-xL ratio, cleaved PARP, apoptotic nuclei visualized by DAPI staining, and COMET formation. In addition, markers of autophagy were measured. Acridine orange staining revealed increased accumulation of autophagic vacuoles (AVs) after QC treatment in a dose-dependent manner in parental cells, and decreased staining in isogenic HCT-116 p53-/- and HCT-116 p21-/- cells. Immunofluorescence of LC3B was significantly lowered in QC-treated cells lacking p53 or p21, compared to the parental cells. Interestingly, the expression of the autophagy marker LC3B-II after exposure to QC was decreased in either p53 or p21 null cells compared to parental cells. After deletion of p21 in HCT-116 p53-/- cells, no change in LC3B-II expression was noted following QC treatment. Collectively, the results suggest that QC-mediated autophagy and apoptosis dependent on p53 and p21.

We previously reported that quinacrine (QC) has anticancer activity against breast cancer cells. Here, we examine the mechanism of action of QC and its ability to inhibit Wnt-TCF signaling in two independent breast cancer cell lines. QC altered Wnt-TCF signaling components by increasing the levels of adenomatous polyposis coli (APC), DAB2, GSK-3β and axin and decreasing the levels of β-catenin, p-GSK3β (ser 9) and CK1. QC also reduced the activity of the Wnt transcription factor TCF/LEF and its downstream targets cyclin D1 and c-MYC. Using a luciferase-based Wnt-TCF transcription factor assay, it was shown that APC levels were inversely associated with TCF/LEF activity. Induction of apoptosis and DNA damage was observed after treatment with QC, which was associated with increased expression of APC. The effects induced by QC depend on APC because the inhibition of Wnt-TCF signaling by QC is lost in APC-knockdown cells, and consequently, the extent of apoptosis and DNA damage caused by QC is reduced compared with parental cells. Because we previously showed that QC inhibits topoisomerase, we examined the effect of another topoisomerase inhibitor, etoposide, on Wnt signaling. Interestingly, etoposide treatment also reduced TCF/LEF activity, β-catenin and cyclin D1 levels commensurate with induction of DNA damage and apoptosis. Lycopene, a plant-derived antioxidant, synergistically increased QC activity and inhibited Wnt-TCF signaling in cancer cells without affecting the MCF-10A normal breast cell line. Collectively, the data suggest that QC-mediated Wnt-TCF signal inhibition depends on APC and that the addition of lycopene synergistically increases QC anticancer activity.

Resveratrol (Res) can modulate multiple cellular pathways relevant for tumorigenesis but is less effective in colon cancer compared to breast cancer. To increase the chemopreventive potential of Res in combination with 5-fluorouracil (5-FU), a systematic study was carried out in colon cancer cells. HCT-116 cells were treated with Res and 5-FU and several cell-based assays, such as MTT, clonogenic, wound healing, DAPI, comet assay, and Western blot, were performed. A significant inhibition of cell proliferation, migration, and increased apoptosis were observed when moderate concentration of Res (15 microM) was associated with very low concentration of 5-FU (0.5 microM). This combination caused apoptosis by blocking the cells at S phase and enhanced the DNA damage. Expression levels of p-JNK and p-p38 were increased without affecting pERK. 5-FU could be used as a therapeutic modality to improve efficacy of Res-based chemotherapy against colon cancer.

On the basis of structures of known topoisomerase II catalytic inhibitors and initial molecular docking studies, bicyclic N-fused aminoimidazoles were predicted as potential topoisomerase II inhibitors. They were synthesized by multicomponent reactions and evaluated against human topoisomerase IIα (hTopoIIα) in decatenation, relaxation, cleavage complex, and DNA intercalation in vitro assays. Among 31 compounds of eight different bicyclic scaffolds, it was found that imidazopyridine, imidazopyrazole, and imidazopyrazine with suitable substituents exhibited potent inhibition of catalytic activity of hTopoIIα while not showing DNA intercalation. Molecular docking studies and molecular dynamics (MD) simulation analysis, ATPase-kinetics and ATP-dependent plasmid relaxation assay revealed the catalytic mode of inhibition of the title compounds plausibly by blocking the ATP-binding site. N-Fused aminoimidazoles showed potent anticancer activities in kidney and breast cancer cell lines, low toxicity to normal cells, relatively higher potency compared to etoposide and 5-fluorouracil in kidney cancer cell lines, and potent inhibition in cell migration. These compounds were found to exert apoptotic effect in G1/S phase.

Most current anti-cancer therapeutic drugs are targeting the proliferation and/or survival of cancer cells while very few drugs are aimed at specifically targeting the dissemination process. Several reports have demonstrated that low-levels or lack of Wnt-5a protein expression in primary breast-, colon-, and prostate cancer tissues correlates with shortened patient recurrence-free survival and overall survival pointing to a biological role of Wnt5a signaling in the dissemination process of cancer cells. The Wnt5a ligand mediates its effects vid interaction with G-protein coupled Frizzled receptors and tyrosine-kinase coupled receptors such as ROR1 and ROR2. Therefore, we developed two peptides, one being a Wnt-5a agonist (Foxy-5) and one being a Wnt-5a antagonist (Box-5). The Foxy-5 is a formylated Wnt5a-derived hexapeptide that mimics the ability of the Wnt-5a molecule to impair cancer cell migration in vitro and significantly reduces the formation of distant metastases in vivo in mouse models of breast- and prostate cancer. In addition, a 4-week toxicology study in rats and dogs with a final dose well exceeding the dose previously used in the mouse models showed no drug-induced toxic reactions.Based on all these pre-clinical data we initiated a clinical phase 1 study with the primary objective to evaluate the safety and tolerability of Foxy-5. All eligible patients are pre-screened for Wnt-5a immunoreactivity in archival tumor tissue and from dose level 7 and onwards only patients with negative or low level Wnt5a expressing metastatic breast-, colon-, or prostate cancer are enrolled in the study. This study has currently recruited cohorts 1-7 without reaching MTD and the final recruitment for the last dose level (dose level 8) is ongoing. Clinical trial information: NCT02020291. This study will be finalized during early fall 2015 and followed by a phase 1b study which will continue dose escalation but with a specific focus on determining the Biological Active Dose (BAD) since Foxy-5 does not possess anti-proliferative activities and is therefore not expected to induce tumor regression. In contrast to breast, colon and prostate cancer, Wnt5a signaling promotes tumor progression in vitro in melanoma and gastric cancer. The antagonistic Wnt5a-derived peptide Box5 possesses the capacity to impair Wnt5a signaling and migration in melanoma cells. We are now performing additional pre-clinical experiments to enable us to test the in vivo effects of Box5 in a melanoma animal model. Future clinical development plans include a phase II study enrolling patients with stage III colorectal cancer and then add Foxy-5 to standard 5FU plus oxaliplatin adjuvant treatment. Citation Format: Tommy Andersson, Lena Axelsson, Purusottam Mohapatra, Chandra Prasad, Peter Grundtvig Soerensen, Morten Mau-Soerensen, Ulrik Lassen, Tine Molvadgaard, Ulla Buhl, Nils Brünner, Dorte Nielsen. Targeting the Wnt-5a signaling pathway as a novel anti-metastatic therapy. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2015 Nov 5-9; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2015;14(12 Suppl 2):Abstract nr A116.

We previously reported that quinacrine (QC) has anticancer activity against breast cancer cells. Here, we report the mechanism of action of QC and its ability to inhibit Wnt-TCF signaling in two independent breast cancer cell lines. QC altered Wnt-TCF signaling components by increasing the levels of adenomatous polyposis coli (APC), DAB2, GSK-3β and axin and decreasing the levels of β-catenin, p-GSK3β (ser 9) and CK1. QC also reduced the activity of the Wnt transcription factor TCF/LEF and its downstream targets cyclin D1 and c-MYC. Using a luciferase-based Wnt-TCF transcription factor assay, it was shown that APC levels were inversely associated with TCF/LEF activity. Induction of apoptosis and DNA damage was observed after treatment with QC, which was associated with increased expression of APC. The effects induced by QC depend on APC because the inhibition of Wnt-TCF signalling by QC is lost in APC-knockdown cells, and consequently, the extent of apoptosis and DNA damage caused by QC was reduced compared with parental cells. Further, previously we have shown that QC inhibits topoisomerase, therefore we tested the effect of another topoisomerase inhibitor, etoposide, on Wnt signaling. Interestingly, etoposide treatment also reduced TCF/LEF activity, β-catenin and cyclin D1 levels commensurate with induction of DNA damage and apoptosis. Lycopene, a plant-derived antioxidant, synergistically increased QC activity and inhibited Wnt-TCF signaling in cancer cells without affecting the MCF-10A normal breast cell line. Collectively, the data suggests that QC-mediated Wnt-TCF signal inhibition depends on APC and that the addition of lycopene synergistically increases QC anticancer activity. Note: This abstract was not presented at the meeting. Citation Format: Ranjan Preet, Rajasubramaniam Shanmugam, Purusottam Mohapatra, Dipon Das, Shakti R. Satapathy, Michael D. Wyatt, Chanakya N. Kundu. Lycopene synergistically enhances quinacrine action to inhibit Wnt-TCF signaling in breast cancer cells through APC. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 2777. doi:10.1158/1538-7445.AM2014-2777

To study the mechanism of 5-fluorouracil (5-FU) resistance in colon cancer cells and to develop strategies for overcoming such resistance by combination treatment.We established and characterized a 5-FU resistance (5-FU-R) cell line derived from continuous exposure (25 μmol/L) to 5-FU for 20 wk in 5-FU sensitive HCT-116 cells. The proliferation and expression of different representative apoptosis and anti-apoptosis markers in 5-FU sensitive and 5-FU resistance cells were measured by the MTT assay and by Western blotting, respectively, after treatment with Resveratrol (Res) and/or 1,3-Bis(2-chloroethyl)-1-nitrosourea (BCNU). Apoptosis and cell cycle arrest was measured by 4',6'-diamidino-2-phenylindole hydrochloride staining and fluorescence-activated cell sorting analysis, respectively. The extent of DNA damage was measured by the Comet assay. We measured the visible changes in the DNA damage/repair cascade by Western blotting.The widely used chemotherapeutic agents BCNU and Res decreased the growth of 5-FU sensitive HCT-116 cells in a dose dependent manner. Combined application of BCNU and Res caused more apoptosis in 5-FU sensitive cells in comparison to individual treatment. In addition, the combined application of BCNU and Res caused a significant decrease of major DNA base excision repair components in 5-FU sensitive cells. We established a 5-FU resistance cell line (5-FU-R) from 5-FU-sensitive HCT-116 (mismatch repair deficient) cells that was not resistant to other chemotherapeutic agents (e.g., BCNU, Res) except 5-FU. The 5-FU resistance of 5-FU-R cells was assessed by exposure to increasing concentrations of 5-FU followed by the MTT assay. There was no significant cell death noted in 5-FU-R cells in comparison to 5-FU sensitive cells after 5-FU treatment. This resistant cell line overexpressed anti-apoptotic [e.g., AKT, nuclear factor κB, FLICE-like inhibitory protein), DNA repair (e.g., DNA polymerase beta (POL-β), DNA polymerase eta (POLH), protein Flap endonuclease 1 (FEN1), DNA damage-binding protein 2 (DDB2)] and 5-FU-resistance proteins (thymidylate synthase) but under expressed pro-apoptotic proteins (e.g., DAB2, CK1) in comparison to the parental cells. Increased genotoxicity and apoptosis were observed in resistant cells after combined application of BCNU and Res in comparison to untreated or parental cells. BCNU increased the sensitivity to Res of 5-FU resistant cells compared with parental cells. Fifty percent cell death were noted in parental cells when 18 μmol/L of Res was associated with fixed concentration (20 μmol/L) of BCNU, but a much lower concentration of Res (8 μmol/L) was needed to achieve the same effect in 5-FU resistant cells. Interestingly, increased levels of adenomatous polyposis coli and decreased levels POL-β, POLH, FEN1 and DDB2 were noted after the same combined treatment in resistant cells.BCNU combined with Res exerts a synergistic effect that may prove useful for the treatment of colon cancer and to overcome drug resistance.

Wheat cv. Warimba plants were grown in a controlled environment in either a long (16 h) or short (8 h) photoperiod. When the apices reached the stage of floret initiation in the long photoperiod, some plants were transferred to the short photoperiod at the same photon irradiance and others to a lower photon irradiance in the long photoperiod. The plants growing initially in the short photoperiod were transferred at a slightly earlier stage of development (lemma initiation) to either the high photon irradiance-long photoperiod environment or to the short photoperiod at the lower photon irradiance. Apices were divided into upper and lower portions and their dry weights and soluble sugar contents were determined. Sucrose alone was found in most of the apices with trace amounts of glucose and fructose being found late in development in the apices of plants growing in the long photoperiod. There was always a higher sucrose concentration in the lower portion of the apex than in the upper. Transfer to a low photon irradiance reduced apical sucrose content, sucrose initially disappearing completely from the upper region of the apices growing in the long photoperiod. There was some subsequent recovery, however. Transfer to the alternative photoperiod also reduced apical sucrose content, although not as severely.

The effects of high temperature on the growth and development of the shoot apex of wheat, cv. Warimba, growing in a short photoperiod (8 h) were examined. High temperature (30°C) from germination onwards delayed the initiation of double ridges on the apex in comparison to low temperature (20°C) but did not affect the subsequent rate of spikelet development to stamen initiation. The rate of primordium production on the apex was reduced at the higher temperature and there was a decrease in the final number of spikelets produced. The growth in dry weight of both the shoot and apex was reduced at the higher temperature, but the relative growth rate of the apex was unaffected after double-ridge initiation. The sucrose concentration in the shoot apex tended to be higher at the higher temperature and so it was concluded that the effects of exposure to high temperature on apex growth were not mediated through effects on assimilate supply.