After cardiovascular diseases, cancer is the leading cause of death in Germany. In 2007, cancer led to around 7.9 million deaths worldwide, accounting for about 13 per cent of all deaths. It is anticipated that as society grows ever older there will be a further increase in cancer-related deaths. However, the average rate of cure has remained unchanged for 20 years. Therefore, the development of new strategies for the treatment of cancer is of great interest. At the University of Konstanz, Dr. Marilena Manea and her research group are working on the development of bioconjugates for targeted cancer chemotherapy. The researchers’ goal is to improve the efficiency and tolerability of the chemotherapeutic drugs that are already being used in hospitals.
Despite the huge effort to develop novel anti-cancer drugs, chemotherapy is still one of the most commonly used treatment strategies. It has been shown that chemotherapy is frequently the only option for treating metastases and very big tumours. However, conventional chemotherapy has quite a number of disadvantages, including the unselective effect on all rapidly dividing cells which can lead to hair loss, nausea and heart muscle damage. In addition, classical cytostatic drugs are quickly metabolised, with the result that higher doses are needed. This in turn increases the number of or exacerbates the adverse reactions of the drug. It has also been shown that cancer cells become resistant to chemotherapy, which again requires the administration of stronger drugs.
Targeted chemotherapy has the potential to overcome the disadvantage of free cytostatic drugs. The objective of targeted chemotherapy is to damage cancerous cells only, sparing healthy cells and hence drastically reducing the adverse reactions of the drugs. "Targeted chemotherapy requires the presence of receptors or other target molecules on the cell surface. In addition, these receptors should only be expressed by cancer cells," said Dr. Manea.
One example of such molecules is the receptors of the gonadotropin-releasing hormone (GnRH) which are highly expressed on many types of cancer cells, compared to their expression in healthy tissues. "Therefore, the GnRH peptide or its analogues can be used as carriers for cytostatic drugs," said Dr. Manea. The receptor-mediated uptake of a hybrid compound consisting of a GnRH and cytostatic drug, which is referred to as bioconjugate, only destroys the cancer cells that express the GnRH receptors. Healthy cells do not take up the bioconjugate and remain intact. "The specificity of these drugs leads to a higher concentration of chemotherapeutic agents in the tumour, which makes the chemotherapy considerably more efficient at the same time as contributing to a reduction in adverse drug effects," explains Marilena Manea. The bioconjugates must be stable in the blood stream, and then be cleaved in the lysosomes once they have been taken up by the cancer cells.The peptide hormone GnRH has a major physiological role in controlling reproduction. The release of the gonadotropic hormones LH (luteinizing hormone) and FSH (follicle stimulating hormone) triggers the secretion of sex steroids. "This endocrine effect of GnRH and its analogues is not wanted in tumour therapy since sex steroids frequently stimulate the tumor growth,, particularly tumours of the reproductive tract," said the researcher from Konstanz. Thus, the development of new GnRH analogues is focused on minimising the hormonal effect at the same time as increasing the anti-tumour activity. A promising GnRH analogue was isolated from the sea lamprey (Petromyzon marinus) and is known as GnRH-III. "It has been shown that this GnRH analogue binds to GnRH receptors and has a lower endocrine effect than human GnRH and its analogues," said Dr. Marilena Manea. In addition, GnRH-III has been shown to have an antiproliferative effect. These characteristics indicate that GnRH-III is a promising carrier molecule for chemotherapeutic drugs, and makes targeted chemotherapy possible.
The development of targeted chemotherapeutic agents involves the coupling of different cytostatic drugs (e.g., daunorubicin, doxorubicin, methotrexate or camptothecin) to the GnRH-III molecule. "Once the bioconjugate has bound to the GnRH receptor, it is internalised by the cell and degraded in the lysosomes," said the researcher. The efficacy of the bioconjugates depends on numerous factors, including the type of cytostatic drug used or the chemical linkage between GnRH-III and the cytostatic drug. Dr. Marilena Manea and her team are investigating the influence of different GnRH-III derivatives, chemical linkages between the drug under investigation and GnRH-III and the use of different cytostatic drugs on in vitro stability and cellular uptake as well as on the anti-cancer effect of the bioconjugates.The team from Konstanz synthesises the bioconjugates by a combination of solid phase peptide synthesis and different chemical ligation methods. Since it is of crucial importance to know the bioconjugates' purity and molecular composition for the correct determination of anti-cancer effect, the bioconjugates are purified by high-pressure liquid chromatography, analysed with by mass spectrometry and their secondary structure is determined using circular dichroism spectroscopy. An important aspect in the therapeutic application of the bioconjugates is their enzymatic stability, e.g. in human serum, lysosomal preparations and in the presence of certain enzymes such as cathepsin B. "When the drug is administered to the patients, care must be taken to ensure that it remains stable while it is transported in the blood in order to prevent the early cleavage of the cytostatic drug from the carrier and the onset of severe side effects," said the chemist. When the drugs are given orally the researchers need to assess the effect that different digestive enzymes in the stomach and intestines might have on the bioconjugates.
Dr. Manea and her team use flow cytometry and fluorescence microscopy to investigate the cellular uptake of the synthesised bioconjugates. They have been able to show that all bioconjugates are taken up by the cells. "The drugs' cytostatic effect was determined by MTT assay (MTT: 3-(4,5-dimethylthiazole-2-yl)-2,5-diphenyltetrazolium bromide) on different human cancer cell lines, such as the MCF-7 breast cancer cell line, the HT-29 colon cancer line and the LNCaP prostate cancer cell line," said Marilena Manea. Initial experiments involving colon carcinoma bearing mice have already shown that the different bioconjugates are highly specific and efficient in vivo.Another approach to increase the treatment efficacy involves the development of bioconjugates that contain more than one cytostatic drug. "Since the number of GnRH receptors on the surface of cancer cells is limited, we assume that multifunctional bioconjugates are able to increase the therapeutic efficiency of the drugs," said Dr. Marilena Manea. Such complex compounds, which contain two identical or different chemotherapeutic agents (e.g., daunorubicin, methotrexate) have been developed and their cytostatic effect has been shown to be greater than that of monofunctional bioconjugates.The researchers still have a long way to go before they develop an effective anti-cancer drug, but Marilena Manea points out that there are quite a few promising candidates and that the findings of her research group provide a "basis for the further development of hybrid drugs with improved stability in serum and increased anti-tumour activity."
Marilena Manea studied chemistry and physics at the Al.I.Cuza University in Iasi, Romania. After a one-year research stay at the University of Konstanz in the Laboratory of Analytical Chemistry and Biopolymer Structure Analysis, she received her PhD in 2006 for her work on "design, structural and immuno-analytical properties of antigenic polypeptides comprising a ß-amyloid-plaque specific epitope" during which she also developed combined B- and T-cell epitope-peptide bioconjugates as new lead structures for vaccines against Alzheimer's disease. After several research stays at the Hungarian Academy of Sciences, Marilena Manea became a fellow of the Zukunftskolleg and junior research group leader at the University of Konstanz, Department of Chemistry, in 2008. She is currently working mainly on targeted cancer chemotherapy (in particular receptor-mediated tumor targeting), synthetic peptide-based vaccines and protein structure analyses.
Dr. Marilena Manea University of KonstanzFuture College and Faculty of ChemistryAnalytical Chemistry and Biopolymer Structure AnalysisTel.: +49(0)7531 / 88-2285E-mail: marilena.manea(at)uni-konstanz.de