Evaluation of ergosterol and its metabolites as LXR agonists and their anticancer potential in colon cancer

Purpose: Aberrant cholesterol homeostasis is a well-recognized hallmark of cancer and implicated in metastasis and chemotherapeutic resistance, the two major causes of cancer associated mortality. Liver X receptors (LXRs) are the key transcription factors that induce cholesterol efflux via enhancing the expression of ABCA1 and ABCG1. Methods: Molecular docking and dynamic simulation studies were done to assess the binding affinity and stability of the receptor ligand complexes. Activation of LXRs was evaluated using the luciferase reporter assay. qRT-PCR and western blotting was done to analyse the mRNA and protein expression of cholesterol homeostasis genes. Flow cytometric analysis was carried out to evaluate the surface expression of ABCA1. The effect of selected sterols on viability of three cancer cell lines and one normal epithelial cell line was assessed using MTT assay. Results: Ergosterol (Erg), ergosta-7,22,24(28)-trien-3β-ol (Erg1), ergosta-5,22,25-trien-3-ol (Erg2), ergosta-5,7,22,24(28)-tetraen-3β-ol (Erg3), and ergosta-7,22-dien-3β-ol (Erg4) displayed good binding affinities and formed stable complexes with both isoforms of LXRs. Treatment with Erg led to 2.5 fold while Erg2 and Erg4 led to 1.7 fold increase in LXR activation. Furthermore, a significant increase in mRNA expression of NR1H2, ABCA1, ABCG1 and ApoE was observed upon Erg treatment and it also led to a 25 fold increase in cell surface expression of ABCA1. All of the sterol were selectively toxicity toxic towards colorectal cancer cells but not towards normal epithelial cells. Conclusion: Our findings suggests that ergosterol activates LXRβ and have significant anticancer activity and thus it could be a likely candidate to manage aberrant cholesterol homeostasis associated with colorectal cancer

Working Lives of Management Consultants

This doctoral research focuses on the exploration of the working lives of ‘management consultants’ with an emphasis on how consultants make sense of, interact with and construct their working lives. From a theoretical perspective, this research is situated within the critical management consulting literature that views consulting as a social phenomenon. In this study, sensemaking perspective has been used as a conceptual framework to explore the processes of management consultants’ sensemaking. This research employs a qualitative, interpretive, social‐constructionist perspective by which to understand the lived experiences of management consultants. The core methodology adopted in this study is life history research. In-depth life history interviews were carried out with research participants from leading consulting firms in the UK and Denmark. These interviews also included a visual inquiry based on the photographs/drawings generated by participants. The research findings highlight the multidimensional nature of the lives of ‘management consultants’ and their processes of sensemaking. Three prominent sensemaking processes emerged as part of the research findings: first, the use of differentiation/comparison with other non-consulting professions (outgroup) by management consultants to make sense of their work by equating challenges, downplaying deficiencies and glorifying the paybacks of consulting profession; second, the use of narrative presentations of 'projects’ that point towards iterative cycles of action and interpretation; and, third, the use of ‘metaphors’ to define their practice and open up ways of ‘seeing’ and intervening

Fish oil suppresses cell growth and metastatic potential by regulating PTEN and NF-κB signaling in colorectal cancer.

Homeostasis in eukaryotic tissues is tightly regulated by an intricate balance of the prosurvival and antisurvival signals. The tumor suppressor PTEN (phosphatase and tensin homolog deleted on chromosome 10), a dual-specificity phosphatase, plays a functional role in cell cycle arrest and apoptosis. NF-κB and its downstream regulators (such as VEGF) play a central role in prevention of apoptosis, promotion of inflammation and tumor growth. Therefore, we thought to estimate the expression of PTEN, Poly-ADP-ribose polymerase (PARP), NF-κBp50, NF-κBp65 and VEGF to evaluate the effect of supplementation of fish oil on apoptotic and inflammatory signaling in colon carcinoma. Male wistar rats in Group I received purified diet while Group II and III received modified diet supplemented with FO∶CO(1∶1)&FO∶CO(2.5∶1) respectively. These were further subdivided into controls receiving ethylenediamine-tetra acetic-acid and treated groups received dimethylhydrazine-dihydrochloride (DMH)/week for 4 weeks. Animals sacrificed 48 hours after last injection constituted initiation phase and that sacrificed after 16 weeks constituted post-initiation phase. We have analysed expression of PTEN, NF-κBp50, NF-κBp65 by flowcytometer and nuclear localization of NF-κB by immunofluorescence. PARP and VEGF were assessed by immunohistochemistry. In the initiation phase, animals receiving DMH have shown increased % of apoptotic cells, PTEN, PARP, NF-κBp50, NF-κBp65 and VEGF however in post-initiation phase no significant alteration in apoptosis with decreased PTEN and increased PARP, NF-κBp50, NF-κBp65 and VEGF were observed as compared to control animals. On treatment with both ratios of fish oil in both the phases, augmentation in % of apoptotic cells, decreased PTEN, PARP, NF-κBp50, NF-κBp65 and VEGF were documented with respect to DMH treated animals with effect being more exerted with higher ration in post-initiation phase. Hence, fish oil activates apoptosis, diminishes DNA damage and inhibits inflammatory signalling in a dose and time dependent manner so as to inhibit progression of colon cancer

Alterations in Lipid Mediated Signaling and Wnt/β-Catenin Signaling in DMH Induced Colon Cancer on Supplementation of Fish Oil

Ceramide mediates inhibition of cyclooxygenase-2 (COX-2) which catalyzes formation of prostaglandin further activating peroxisome proliferator-activated receptorγ (PPARγ) and Wnt/β-catenin pathway; and hence plays a critical role in cancer. Therefore, in current study, ceramide, COX-2, 15-deoxy prostaglandin J2(15-deoxy PGJ2), PPARγ, and β-catenin were estimated to evaluate the effect of fish oil on lipid mediated and Wnt/β-catenin signaling in colon carcinoma. Male Wistar rats in Group I received purified diet while Groups II and III received modified diet supplemented with FO : CO(1 : 1) and FO : CO(2.5 : 1), respectively. These were further subdivided into controls receiving ethylenediaminetetraacetic acid and treated groups receiving dimethylhydrazine dihydrochloride (DMH)/week for 4 weeks. Animals sacrificed 48 hours after last injection constituted initiation phase and those sacrificed after 16 weeks constituted postinitiation phase. Decreased ceramide and increased PPARγ were observed in postinitiation phase only. On receiving FO+CO(1 : 1)+DMH and FO+CO(2.5 : 1)+DMH in both phases, ceramide was augmented whereas COX-2, 15-deoxy PGJ2, and nuclear translocation of β-catenin were reduced with respect to cancerous animals. Decrease was more significant in postinitiation phase with FO+CO(2.5 : 1)+DMH. Treatment with oils increased PPARγ in initiation phase but decreased it in postinitiation phase. Hence, fish oil altered lipid mediated signalling in a dose and time dependent manner so as to inhibit progression of colon cancer

Effect of supplementation of fish oil on nuclear localization of NF-κBp65 in initiation and post-initiation phase.

<p>The isolated fixed colonocytes were permeabilized and incubated with monoclonal antibody against NF-κB p65 and corresponding FITC conjugated secondary antibody The sections were counterstained with PI to visualise nuclear localisation. Red fluorescence represents the nuclear staining by PI with no expression of NF-κB (depicted by “dotted arrow”) and green fluorescence indicates the expression of NF-κB p65. Yellow colour indicates the localisation of NF-κB p65 from cytoplasm to nucleus (represented by “arrow”). A–D depicts the initiation phase and E–H represents the post-initiation phase. (A and E) Control, (B and F) DMH treated, (C and G) FO+CO(1∶1)+DMH, (D and H) FO+CO(2.5∶1)+DMH (Magnification 400X).</p

Inhibition of PARP activity on receiving different ratios of fish oil and corn oil in experimentally induced colon cancer.

<p>Immunohistochemical staining of PARP in formalin fixed paraffin embedded sections of colon tissue. A–D depicts the representative photomicrographs of initiation phase and E–H represents the post-initiation phase (“arrow” depicts the expression of PARP). (A and E) Control, (B and F) DMH treated, (C and G) FO+CO(1∶1)+DMH, (D and H) FO+CO(2.5∶1)+DMH (Magnification 400X).</p

Alterations in VEGF expression on administration of both the ratios of fish oil and corn oil.

<p>The expression of VEGF was analyzed using immunohistochemistry in formalin fixed paraffin embedded sections of colon tissue. A–D depicts the representative photomicrographs of initiation phase and E–H represents the post-initiation phase (→ depicts the expression of VEGF). (A and E) Control, (B and F) DMH treated, (C and G) FO+CO(1∶1)+DMH, (D and H) FO+CO(2.5∶1)+DMH (Magnification 400X).</p

Effect of fish oil and corn oil on NF- κB p65 expression.

<p>The results are expressed as Mean±S.D. for n = 8.</p><p>p<0.001 wrt control group,</p><p>^### p<0.001 wrt DMH treated,</p><p>p<0.001 wrt FO+CO(1∶1)+DMH.</p

Effect of different ratios of fish oil and corn oil on % of apoptotic cells in the initiation phase of experimentally induced colon cancer.

<p>The isolated colonocytes were incubated with Hoechst 342 (H342) dye and PI. Photomicrographs were acquired using fluorescent microscope (Nikon Eclipse 80<i>i</i>) A- control group, B- DMH treated, C) FO+CO(1∶1)+DMH, D) FO+CO(2.5∶1)+DMH. Dark blue colour represents the normal live cells, faint blue or pink represents apoptotic cells (Magnification 400×). E) Graphical representation of % of live and apoptotic cells in different groups. The results are expressed as Mean±S.D. for n = 4 (^**p<0.01 wrt control, ^###p<0.001 wrt DMH).</p

Alterations in the % of apoptotic cells on administration of both the ratios of fish oil and corn oil in post-initiation phase.

<p>The isolated colonocytes were incubated with Hoechst 342 (H342) dye and PI. Photomicrographs were acquired using fluorescent microscope (Nikon Eclipse 80<i>i</i>) A- control group, B- DMH treated, C) FO+CO(1∶1)+DMH, D) FO+CO(2.5∶1)+DMH. Dark blue colour represents the normal live cells, faint blue or pink represents apoptotic cells (Magnification 400×). E) Graphical representation of % of live and apoptotic cells in different groups. The results are expressed as Mean±S.D. for n = 4 (^###p<0.001 wrt DMH).</p