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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.

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.

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.

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.

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.