Curcumin Chemosensitizes 5-Fluorouracil Resistant MMR-Deficient Human Colon Cancer Cells in High Density Cultures

Objective

Treatment of colorectal cancer (CRC) remains a clinical challenge, as more than 15% of patients are resistant to 5-Fluorouracil (5-FU)-based chemotherapeutic regimens, and tumor recurrence rates can be as high as 50–60%. Cancer stem cells (CSC) are capable of surviving conventional chemotherapies that permits regeneration of original tumors. Therefore, we investigated the effectiveness of 5-FU and plant polyphenol (curcumin) in context of DNA mismatch repair (MMR) status and CSC activity in 3D cultures of CRC cells.

Methods

High density 3D cultures of CRC cell lines HCT116, HCT116+ch3 (complemented with chromosome 3) and their corresponding isogenic 5-FU-chemo-resistant derivative clones (HCT116R, HCT116+ch3R) were treated with 5-FU either without or with curcumin in time- and dose-dependent assays.

Results

Pre-treatment with curcumin significantly enhanced the effect of 5-FU on HCT116R and HCR116+ch3R cells, in contrast to 5-FU alone as evidenced by increased disintegration of colonospheres, enhanced apoptosis and by inhibiting their growth. Curcumin and/or 5-FU strongly affected MMR-deficient CRC cells in high density cultures, however MMR-proficient CRC cells were more sensitive. These effects of curcumin in enhancing chemosensitivity to 5-FU were further supported by its ability to effectively suppress CSC pools as evidenced by decreased number of CSC marker positive cells, highlighting the suitability of this 3D culture model for evaluating CSC marker expression in a close to vivo setting.

Conclusion

Our results illustrate novel and previously unrecognized effects of curcumin in enhancing chemosensitization to 5-FU-based chemotherapy on DNA MMR-deficient and their chemo-resistant counterparts by targeting the CSC sub-population. (246 words in abstract).     

A computational approach to studying monomer selectivity towards the template in an imprinted polymer

A computational approach was proposed to study monomer–template interactions in a molecularly imprinted polymer (MIP) in order to gain insight at the molecular level into imprinting polymer selectivity, regarding complex formation between template and monomer at the pre-polymerisation step. This is the most important step in MIP preparation. In the present work, chlorphenamine (CPA), diphenhydramine (DHA) and methacrylic acid (MAA), were chosen as the template, non-template, and monomer, respectively. The attained complexes were optimised, and changes in the interaction energies, atomic charges, IR spectroscopy results, dipole moment, and polarisability were studied. The effects of solvent on template–monomer interactions were also investigated. According to a survey of the literature, this is the first work in which dipole moment and polarisability were used to predict the types of interactions existing in pre-polymerisation complexes. In addition, the density functional tight-binding (DFTB) method, an approximate version of the density functional theory (DFT) method that was extended to cover the London dispersion energy, was used to calculate the interaction energy.     

A New Self-Loading Locomotion Mechanism for Wall Climbing Robots Employing Biomimetic Adhesives

A versatile locomotion mechanism is introduced and experimentally verified. This mechanism comprises four rectangular wheels (legs) with rotational phase difference which enables the application of pressure to each contacting surface for securing it to the surface using bio-inspired or pressure-sensitive adhesives. In this mechanism, the adhesives are applied to two rigid plates attached to each wheel via hinges incorporating torsional springs. The springs force the plates back to their original position after the contact with the surface is lost in the course of locomotion. The wheels are made of low-modulus elastomers, and the pressure applied during contact is controlled by the elastic modulus, geometry and phase difference of wheels. This reliable adhesion system does not rely upon gravity for adhering to surfaces, and provides the locomotion mechanism with the ability to climb walls and transition from horizontal to vertical surfaces.     

Investigation on functional and numerical responses of Encarsia acaudaleyrodis parasitizing Bemisia tabaci on cucumber

Functional and numerical responses of Encarsia acaudaleyrodis Hayat parasitizing Bemisia tabaci Gennadius were investigated under laboratory conditions. Newly emerged pairs of parasitoids were confined for 24 h in 8 cm Petri dishes together with 5, 10, 20, 40, 60, or 100 individuals of B. tabaci at their third nymphal stage. The parasitoid exhibited a type II functional response, attacking up to 9.6 nymphs when 100 had been provided. The rate of attack and handling time were 0.0554 and 2.3068 h, respectively. The number of eggs deposited by a single parasitoid depended on the number of host individuals, the maximum being 44.4 when the number of nymphs was 100. At the above-mentioned densities, the parasitoid lived 8.5 days on average.     

Spectroscopic studies of the effects of glycation of human serum albumin on L-Trp binding

Modification of proteins by nonenzymatic glycation is one of the underlying factors that contribute to the development of the complications of diabetes. Human serum albumin (HSA) is one of the major targets of interaction with glucose through the Maillard reaction. The effects of 1 and 5 mg/ml glucose concentrations, which are consistent with blood glucose levels found in diabetic patients, on human serum albumin were studied by circular dichroism and fluorescence spectroscopy in sodium phosphate buffer, pH 7.4. Partial denaturation and changes in the structural integrity of HSA are caused by glycation at lower (1 mg/ml) and higher (5 mg/ml) concentrations of glucose. To study the relationship between structure and function, we investigated the interaction of L-tryptophan (L-Trp) with glycated and non-glycated HSA. The results showed that L-Trp, as the only free amino acid that substantially binds to HSA, has a lower affinity for the glycated form (especially at low concentrations of glucose) than for non-glycated HSA.     

Bcr and Abr Cooperate in Negatively Regulating Acute Inflammatory Responses

Bcr and Abr are GTPase-activating proteins for the small GTPase Rac. Both proteins are expressed in cells of the innate immune system, including neutrophils and macrophages. The function of Bcr has been linked to the negative regulation of neutrophil reactive oxygen species (ROS) production, but the function of Abr in the innate immune system was unknown. Here, we report that mice lacking both proteins are severely affected in two models of experimental endotoxemia, including exposure to Escherichia coli lipopolysaccharide and polymicrobial sepsis, with extensive microvascular leakage, resulting in severe pulmonary edema and hemorrhage. Additionally, in vivo-activated neutrophils of abr and bcr null mutant mice produced excessive tissue-damaging myeloperoxidase (MPO), elastase, and ROS. Moreover, the secretion of the tissue metalloproteinase MMP9 by monocytes and ROS by elicited macrophages was abnormally high. In comparison, ROS production from bone marrow monocytes was not significantly different from that of controls, and the exocytosis of neutrophil secondary and tertiary granule products, including lactoferrin, was normal. These data show that Abr and Bcr normally curb very specific functions of mature tissue innate immune cells, and that each protein has distinct as well as partly overlapping functions in the downregulation of inflammatory processes.BCR originally was discovered as a human gene on chromosome 22 that, in chronic myeloid leukemia, becomes fused to the c-ABL tyrosine kinase gene originating from chromosome 9 (18). The normal gene encodes a 160-kDa protein that contains a domain with GTPase-activating (GAP) activity toward Rho family GTPases (7, 11, 12, 32, 36). There is only one other gene in mouse and human, called ABR, that is closely homologous to BCR (17). Abr shares several domains with Bcr, which includes a Dbl homology (DH) domain and a GAP domain. Bcr has an additional N-terminal part consisting of a coiled-coil and a serine/threonine kinase domain that is not present in Abr, suggesting that each GAP has a distinct cellular function.Rho GTPases, including Rho, Rac, and Cdc42, play important roles in many functions of cells of the innate immune system (16). They cycle between active GTP and inactive GDP-bound conformations. GAP proteins catalyze the conversion of bound GTP to GDP on Rho GTPases and thus act as negative, inactivating regulators.In previous studies, we showed that both Abr and Bcr specifically act as GAPs for Rac and not for the related Cdc42 (6). To investigate the normal cellular function of these two related GAPs, we generated mice defective in the production of Abr or Bcr through gene targeting. Mice that lack both proteins have defects in the architecture of the inner ear, with the partial absence of otoconia and hair cells. Additionally, postnatal cerebellar development is abnormal, with a persistence of ectopic granule cells at the cerebellar surface. These combined abnormalities cause persistent circling and balance problems (20, 21).As reported previously, neutrophils from mice lacking Bcr produce increasing amounts of reactive oxygen species (ROS), and bcr^−/− mice injected with Escherichia coli lipopolysaccharide (LPS) are much more severely affected than are wild-type mice (39). We further explored the role of Bcr and Abr in the innate immune system with a detailed study of bone marrow-derived macrophages (BMM). Interestingly, macrophages isolated from double-knockout (abr × bcr^−/−) mice exhibited multiple defects. These include aberrant actin cytoskeletal organization and the increased colony-stimulating factor 1-stimulated chemotaxis and phagocytosis of opsonized zymosan or E. coli (6).In the current study, we examined whether the defects observed in vitro result in an observable phenotype in vivo, under inflammatory conditions. Here, we report that Abr plays a distinct role in negatively regulating the innate immune system in vivo, as well as exhibiting overlap with the function of Bcr. Mice lacking both Abr and Bcr have a severely impaired ability to resolve septic shock, showing that the activity of both proteins is required for the appropriate negative control of innate immune responses.     

Stabilized β-catenin in lung epithelial cells changes cell fate and leads to tracheal and bronchial polyposis

The precise mechanisms by which β-catenin controls morphogenesis and cell differentiation remain largely unknown. Using embryonic lung development as a model, we deleted exon 3 of β-catenin via Nkx2.1-cre in the Catnb[+/lox(ex3)] mice and studied its impact on epithelial morphogenesis. Robust selective accumulation of truncated, stabilized β-catenin was found in Nkx2.1-cre;Catnb[+/lox(ex3)] lungs that were associated with the formation of polyp-like structures in the trachea and main-stem bronchi. Characterization of polyps suggests that accumulated β-catenin impacts epithelial morphogenesis in at least two ways. “Intracellular” accumulation of β-catenin blocked differentiation of spatially-appropriate airway epithelial cell types, Clara cells, ciliated cells and basal cells, and activated UCHL1, a marker for pulmonary neuroendocrine cells. There was also evidence for a “paracrine” impact of β-catenin accumulation, potentially mediated via activation of Bmp4 that inhibited Clara and ciliated, but not basal cell differentiation. Thus, excess β-catenin can alter cell fate determination by both direct and paracrine mechanisms.     

Cytokine response following transthoracic and transhiatal esophagectomy in patients with esophageal cancer

Curative esophageal resection is usually performed using either a transthoracic (TT) or transhiatal (TH) approach. Forty patients with esophageal squamous cell carcinoma who underwent esophagectomies (24 TT and 16 TH), 12 patients who underwent surgery for gastric cancer, and 16 healthy individuals were enrolled in this study. Blood samples were taken from the patients, pre- and post-surgery. The levels of synthesis of T-helper 1 and 2 cytokines were assessed in vitro in the presence of mitogen. Our initial data indicated that at admission, 24 h before surgery, blood cells from both groups of esophageal cancer patients produced significantly lower levels of the Th1 cytokines, IFN-gamma and IL-2 than those from cells of healthy donors. Cells collected from gastric cancer patients prior to surgery produced intermediate levels of IFN-gamma and IL-2: significantly lower than healthy donors, and slightly more (non-significant) than esophageal cancer patients. These results contrast with those for the production of Th2 cytokines prior to surgery, which did not differ significantly between any groups: either the esophageal or gastric cancer patients, or healthy donors. Th1 and Th2 cytokine production was then studied using blood cells collected seven days after surgery. Cells from both groups of esophageal cancer patients, undergoing either TT or TH surgery, produced significantly lower levels of the Th1 cytokines, IFN-gamma and IL-2 than those from cells of gastric cancer patients who had undergone surgery. Postoperative and preoperative production was compared. For patients who had undergone TT esophageal resection, we observed that the post-operative production of IL-2, IL-5 and IL-13 was significantly lower than the pre-operative production of those cytokines. Such reduced post-operative compared to pre-operative production was only significant in patients who had undergone TT esophagectomy. A similar, but non-significant trend was observed in patients who had undergone either TH esophagectomy, or gastrectomy. The results indicate that digestive tract cancer patients, both esophageal and gastric, have (prior to surgery), a significantly reduced, basal, mitogen-induced production of Th1 but not of Th2 cytokine. Post-operatively, a significantly reduced production of Th1 and Th2 cytokines, except for IFN-gamma, was observed only in patients who had undergone surgical esophageal resection using the TT method.     

Engineering characterisation of a single well from 24-well and 96-well microtitre plates

The detailed engineering characterisation of shaken microtitre-plate bioreactors will enhance our understanding of microbial and mammalian cell culture in these geometries and will provide guidance on the scale-up of microwell results to laboratory and pilot scale stirred bioreactors. In this work computational fluid dynamics (CFD) is employed to provide a detailed characterisation of fluid mixing, energy dissipation rate and mass transfer in single well bioreactors from deep square 24-well and 96-well microtitre plates. The numerical predictions are generally found to be in good agreement with experimental observation of the fluid motion and measured values of the key engineering parameters. The CFD simulations have shown that liquid mixing is more intensive in 96-well than in 24-well bioreactors due to a significant axial component to the fluid velocity. Liquid motion is strongly dependent on the orbital shaking amplitude which generally has a greater impact than the shaking frequency. Average power consumptions of 70–100 W m⁻³ and 500–1000 W m⁻³, and overall mass transfer coefficient, k_La, values of 0.005–0.028 s⁻¹ and 0.056–0.10 s⁻¹ were obtained for 24-well and 96-well bioreactors respectively at an orbital shaking amplitude of 3 mm and shaking frequencies ranging from 500 rpm to 1500 rpm. The distribution of energy dissipation rates within each bioreactor showed these to be greatest at the walls of the well for both geometries. Batch culture kinetics of E. coli DH5 showed similar maximum specific growth rates and final biomass yields in shaken 24-well and shake flask bioreactors and in stirred miniature and 20 L bioreactors at matched k_La values. The CFD simulations thus give new insights into the local and overall engineering properties of microwell bioreactor geometries and further support their use as high throughput tools for the study and optimisation of microbial and mammalian cell culture kinetics at this scale.