Research Projects

Anticancer agent research – Prof Annie Joubert

Anticancer agent research – Prof Annie Joubert

Prof Annie Joubert

Department of Physiology, University of Pretoria

E-mail: annie.joubert@up.ac.za

Title of the project

In silico analysis, synthesis and in vitro evaluation of novel anticancer agents

Researchers

  • Prof F Joubert, Bioinformatics and Computational Biology Unit, University of Pretoria
  • Dr L Lafanechère, Centre de Criblage pour des Molécules Bio-Actives, CEA/ GRENOBLE, FRANCE
  • M Potgieter, School of Chemistry, University of the Witwatersrand, Johannesburg
  • Dr KH Sippel, Department of Biochemistry and Molecular Biology, McKnight Brain Institute of the University of Florida, College of Medicine, USA
  • Prof R Vleggaar, Professor of Organic Chemistry, Head of the Department of Chemistry, University of Pretoria, Pretoria
  • Drr G Morgans and C Edlin, iThemba Pharmaceuticals (PTY) Ltd, Modderfontein, South Africa

Project description

Microtubules play a central role in mitosis and cell division and are important targets in anticancer drug development. Several anticancer drugs clinically important, including the Vinca alkaloids vinblastine, vincristine, and vinorelbine and the taxanes paclitaxel and docetaxel specifically target tubulin. These compounds (Vinca alkaloids and taxanes) interfere with microtubule dynamics and produce a characteristic mitotic arrest phenotype. Considering the clinical success of these agents, microtubule cytoskeleton represents one of the most highly validated cancer targets identified to date.

Our research involved discovery of compounds that are capable of selectively inhibiting the activity of cancer-associated proteins. This constitutes a main component of drug development research. Research is focused on improving treatments to enable the anticancer drug to affect only the cancer cell and at low dosages with less frequent treatment intervals. In addition, we have designed new and improved estradiol analogues for tubulin-, kinesin motor protein and carbonic anhydrase (CA) (CA II, IX, XII) binding affinity by means of in silico analysis at the Bioinformatics Unit at the University of Pretoria. Since these analogues are not commercially available, they were synthesized by iThemba Pharmaceuticals (PTY) Ltd. In silico and in vitro studies were conducted to elucidate each compound’s signal transduction mechanism and to verify their potential as anticancer agent in vitro, as already been tested. Two of these newly-designed compounds are selective inhibiting a specific protein that contributes towards cancer metastasis and need to be assessed to determine clinical application.

Non-scientific report

The designed anticancer compounds were proven to be five to eight times more potent when compared to 2-methoxyestradiol (2ME). (The latter revealed low oral bioavailability and rapid metabolic degradation.) These demonstrate extremely low concentrations for a drug to have anticancer activity when compared to conventional current treatments. Data are promising and demonstrate a synergistic effect of impacting on cancer cell growth. This research strategy also targets the deregulated metabolism associated with cancers contributing to the fight against cancer.

Aim of the project

The objectives are summarized as the in silico design and analysis, synthesis and investigation of estradiol analogues for tubulin-, kinesin motor protein and carbonic anhydrase (CA) (CA II, IX, XII)) signal transduction mechanisms and the verification of their potential anticancer activity in vitro.

What has been achieved to date?

High resolution mass spectrophotometry of the compounds was conducted at the School of Chemistry, University of the Witwatersrand, Johannesburg in order to determine absolute mass of novel anticancer compounds. High throughput drug binding crystallographic assays for carbonic anhydrase II, IX and XII were conducted at the Department of Biochemistry and Molecular Biology, McKnight Brain Institute of the University of Florida, College of Medicine, University of Florida in the USA. These included crystallization, data collection and structure solution. Oxygen-18 kinetics of carbonic anhydrase II, IX and XII were carried out in order to determine the Ki-inhibition constant of the novel compounds. Our collaborators at the Centre de Criblage pour des Molécules Bio-Actives, Institut de Recherches en Technologies et Sciences pour le Vivant in Grenoble, France conducted tubulin polymerization assays on some of our in silico-designed and synthesized analogues. Data were published in journals listed below. In addition, four manuscripts have been submitted to international accredited peer-reviewed journals in the past 2 months (currently under review).

International accredited peer-reviewed articles for 2010

  1. M.H. Visagie and A.M. Joubert (2010) In vitro effects of 2-methoxyestradiol-bis-sulphamate on cell numbers, membrane integrity, morphology and possible induction of apoptosis and autophagy in a non-tumorigenic breast epithelial cell lineCell. Mol.Biol Lett. 15, 564-581
  2. M.H. Visagie and A.M. Joubert (2010) Immunotherapy and its role in cancer. Biomed. Res. 21(4), 1-5
  3. C. Vorster and A. Joubert (2010) In vitro effects of 2-methoxyestradiol-bis-sulphamate on cell growth, morphology and cell cycle dynamics in the MCF-7 breast adenocarcinoma cell line. Biocell 34(2),71-79
  4. T. Mqoco, S. Marais and A. Joubert Influence of 2-methoxyestradiol-bis-sulphamate on cell growth, morphology and its possible induction of cell death in esophageal carcinoma cells Biocell 34(3): 113-120
  5. C.J.J. Vorster and A.M. Joubert (2010) In vitro effects of 2-methoxyestradiol-bis-sulphamate on the non-tumorigenic MCF-12A cell line Cell Biochem. Funct. 28(5), 412-419
  6. B.A. Stander, S. Marais, C.J. Vorster and A.M. Joubert (2010) In vitro effects of 2-methoxyestradiol on morphology, cell cycle progression, cell death and gene expression changes in the tumorigenic MCF-7 breast epithelial cell line. J. Steroid Biochem.Mol. Biol. 119(3-5), 149-160
  7. B.A. Stander, F. Joubert and A. Joubert Docking, synthesis and in vitro evaluation of antimitotic estrone analogs. Chemical Biology & Drug Design 77, 173-181
  8. M.H. Visagie and A.M. Joubert (2011) 2-Methoxyestradiol-bis-sulphamate induces apoptosis and autophagy in atumorigenic breast epithelial cell line. Molecular and Cellular Biochemistry (in press)

As already mentioned, postgraduate students presented their research at the 22nd Congress of the South African Society for Biochemistry and Molecular Biology in January 2010 in Bloemfontein. They also had the opportunity to present their data at the first South African Cell Death Society conference anticipated in Cape Town in January 2011. Four postgraduate students won prizes for their contribution at the University of Pretoria’sMedical Faculty Day and two of them received research awards for ‘best publication’ and ‘runner-up’ in the ‘Basic Sciences’ category at the Faculty of Health Sciences’s Gala evening. Two of my postgraduate students (BA Stander-PhD student) and X Yu (MSc student) will present their data at the Gordon Research Conference on Cell Growth & Proliferation (June 26 – July 1) at the University of New England. BA Stander will also give an oral presentation at the PSSA conference in August this year (UWC). Eight publications appeared (2010) from my team as listed above.

Training of students:
The following students are being trained in cell culture techniques and maintenance:
1. XX Yu (enrolled for MSc Physiology in 2010), 2. TV Mqoco (enrolled for MSc Physiology in 2010), 3. BA Stander (enrolled for a PhD Human Physiology in 2009), 4. CJJ Vorster (enrolled for MSc Physiology in 2008), 5. MH Visagie (enrolled for PhD in Physiology in 2011), 6. AE Theron (enrolled for MSc in Physiology in 2011), 2 Hons students enrolled in 2011 (S Nkandeu, E Wolmarans).

Training of technical staff at the department of Physiology:
S Alummoottil, D Ntuli and Y Hlope

What do you hope to achieve in the next year?

Broad objective: To evaluate and elucidate the mechanism of action of these newly synthesized antimitotic compounds in order to stimulate future research for in vivo testing in this field.

A summary of the in vitro cellular and molecular studies anticipated to determine the mechanism of action of lead compounds so far identified in this project:

  • Induction of autophagy as another type of cell death (aggresome detection)
  • Measurement of cytochrome c release (Cyto-Light anti-cytochrome c FITC flow cytometry)
  • Measurement of increased cyclin B levels (anti-cyclin B antibody to study the possible induction of apoptosis through a G2/M block (thus with accumulation of cyclin B)
  • Mitochondrial membrane potential measurement via confocal microscopy
  • Measurement of intracellular hydrogen peroxide levels (by employing 2′,7′-dichlorofluorescein diacetate (DCFH-DA) as molecular probe)
  • Immunofluorescence analysis of the compounds’ effects on cellular microtubule network
  • Gene expression profiles, as well as protein arrays employing microarray techniques and bioinformatics analysis
  • Confirmation of microarray results by investigating affected genes further on protein level culminating in unraveling the signal transduction mechanism of each of these analogues with possible anticancer potential
  • Depending on the results obtained appropriate signal transduction assays will be conducted

Peer-reviewed publications

  • M.H. Visagie and A.M. Joubert (2010) In vitro effects of 2-methoxyestradiol-bis-sulphamate on cell numbers, membrane integrity, morphology and possible induction of apoptosis and autophagy in a non-tumorigenic breast epithelial cell lineCell. Mol. Biol Lett. 15, 564-581
  • M.H. Visagie and A.M. Joubert (2010) Immunotherapy and its role in cancer. Biomed. Res. 21(4), 1-5
  • C. Vorster and A. Joubert (2010) In vitro effects of 2-methoxyestradiol-bis-sulphamate on cell growth, morphology and cell cycle dynamics in the MCF-7 breast adenocarcinoma cell line. Biocell 34(2), 71-79
  • T. Mqoco, S. Marais and A. Joubert Influence of 2-methoxyestradiol-bis-sulphamate on cell growth, morphology and its possible induction of cell death in esophageal carcinoma cells Biocell 34(3): 113-120
  • C.J.J. Vorster and A.M. Joubert (2010) In vitro effects of 2-methoxyestradiol-bis-sulphamate on the non-tumorigenic MCF-12A cell line Cell Biochem. Funct. 28(5), 412-419
  • B.A. Stander, S. Marais, C.J. Vorster and A.M. Joubert (2010) In vitro effects of 2-methoxyestradiol on morphology, cell cycle progression, cell death and gene expression changes in the tumorigenic MCF-7 breast epithelial cell line. J. Steroid Biochem. Mol. Biol. 119(3-5), 149-160
  • B.A. Stander, F. Joubert and A. Joubert Docking, synthesis and in vitro evaluation of antimitotic estrone analogs (in press Chemical Biology & Drug Design 77, 173-181
  • M.H. Visagie and A.M. Joubert (2011) 2-Methoxyestradiol-bis-sulphamate induces apoptosis and autophagy in a tumorigenic breast epithelial cell line. Molecular and Cellular Biochemistry (in press)

In addition, three manuscripts have been submitted to international accredited peer-reviewed journals in the past 2 months (currently under review).

Abstracts

  • Postgraduate students participated at the South African Academy of Sciences(October 2010), as well as the 22nd Congress of the South African Society for Biochemistry and Molecular Biology, January 2010, Bloemfontein, South Africa and at the first South African Cell Death Society conference in Cape Town in January 2011
  • Cancer and Metabolism: Pathways to the Future, September 2010, Edinburgh, Scotland
  • Gordon Research Conference on Cell Growth & Proliferation (26 June – 1 July) at theUniversity of New England
  • BA Stander will also present a paper on his PhD research at the PSSA conference in August 2011 at UWC.

Conferences

  • B.A. Stander, F. Joubert and A.M. Joubert (2010) The in vitro effects of new antimitotic molecules in human breast adenocarcinoma cells. South African Journal of Science and Technology (In press)
  • C.J.J. Vorster and A.M. Joubert (2010) In vitro evaluation of 2-methoxyestradiol-bis-sulphamate and Sutherlandia frutescens extracts as possible anticancer agents. South African Journal of Science and Technology 29(4), 211-212
  • T. Mqoco, S. Marais and A. Joubert Influence of 2-methoxyestradiol-bis-sulphamate on cell growth, morphology and its possible induction of cell death in esophageal carcinoma cells 29(4),212
  • MH Visagie, BA Stander and AM Joubert (2010) In vitro cell signalling events of 2-methoxyestradiol-bis-sulphamate in a breast adenocarcinoma- and a non-tumorigenic breast epithelial cell line. South African Journal of Science and Technology 29(4), 229

Oral/poster presentations

  • 22nd Congress of the South African Society for Biochemistry and Molecular Biology, January 2010, Bloemfontein, South Africa
  • Cancer and Metabolism: Pathways to the Future, September 2010, Edinburgh, Scotland
  • South African Academy: Biological Sciences, October 2010, Pretoria, South Africa
  • Gordon Research Conference on Cell Growth & Proliferation (26 June – 1 July) at the University of New England
  • BA Stander will also present a paper on his PhD research at the PSSA conference in August 2011 at UWC

Download booklet

CANSA Detectives – ‘Designing Weapons Against Cancer’ in Silico Analysis, Synthesis and in Vitro Evaluation of Novel Anticancer Agents


« BACK


Do you have a question?