Biological activity of novel synthetic tylophorine analogs in MCF-7 breast cancer cells Przemysław Czajkowski 1, Edyta Andrulewicz 1, Anna Bielawska 2, Krzysztof Bielawski 3, Zbigniew Kałuża 4, Agnieszka Gornowicz 2, Anna Galicka 1 Department of Medical Chemistry1, Department of Biotechnology2, Department of Synthesis and Technology of Drugs3, Institute of Organic Chemistry, Polish Academy of Sciences4

BACKGROUND Breast cancer One of the most common cancers and continues to rank one of the top causes of death in woman The high mortality rate associated with breast cancer is directly related to its ability to readily metastasize

BACKGROUND Cell to ECM adhesion is regulated by specefic cell surface cellular adhesion molecules (CAM) knows as integrins. The integrins transmit mechanical stimuli from the ECM to the intracellular cytoskeleton

BACKGROUND The mechanism of contribution of ECM proteins and their receptors in invasiveness of breast cancers is still not fully understood

COLLAGEN Collagens have as a major function to serve as a structural scaffold in forming tissues. Defects in these structural proteins have severe consequences for tissue integrity Decreased amount of collagen in ECM is known to enhance motility and invasion of neoplastic cells, but it also contribute to inhibition of cell growth and induction of apoptosis Collagens have as a major function to serve as a structural scaffold in forming tissues—being the mold onto which tissues grow. In normal adult tissues, collagens fulfill the function of maintaining tissue structure, but during fibrosis, excess collagen synthesis may cause tissue dysfunction.

INTEGRINS Regulate cell-cell and cell-matrix adhesion and thereby play critical roles in tumor progression and metastasis Upregulated expression in human cancers frequently indicates poor prognosis α2β1 integrin functionally inhibits breast tumor metastasis, and α2 expression may serve as an important biomarker of metastatic potential and patient survival

METALLOPROTEINASES Enzymes, that are capable of degrading all kinds of ECM proteins MMP play a key role in physiological processes such as development, angiogenesis, and also in pathological processes such as tumor invasion and metastasis MMP-2 is a marker in predicting phenotypes of tumor including growth, progression and metastasis Elevated expression of MMP-9 is associated with increased metastatic potential in many cancer types including breast cancer Matrix Metalloproteinases 2 is a key molecule in cellular invasion and metastasis.

AIM OF THE STUDY To assess and evaluate the cytotoxic, cytostatic and biological activity of novel synthetic tylophorine analogs in MCF-7 breast cancer cells

MATERIALS Tylophorine and other alkaloids from Asclepiadacae and Moracae family display a high anticancer activity. However, their pronounced CNS toxicity and other side effects, discouraged further consideration of these alkaloids for drug development Consequently, the synthesis of active analogs devoid of dangerous for patients health side effects is actual of great interest

MATERIALS

MATERIALS GD11 GD9 GD41

METHODS Evaluation of the cytotoxicity of novel synthetic tylophorine analogs (GD9, GD11 and GD41) employing a MTT assay and [3H]thymidine incorporation into DNA Viability and DNA synthesis was demonstrated after 24 hour of incubation with the novel drugs used in different concentrations. The effect of new tylophorine analogs was compared with camptothecin which was used as a reference agent Expression of type I collagen and α2β1 integrin was performed by real-time RT-PCR analysis MMP activity was detected by zymography

RESULTS

RESULTS VIABILITY OF CELLS All new synthetic tylophorine analogs exhibited higher cytotoxic activity against the investigated breast cancer cells than camptothecin. The most effective was GD41 with IC50 value 25 µM.

RESULTS DNA SYNTHESIS Similarly, all of tested agents inhibited cell proliferation. GD41 and GD9 were stronger inhibitors of DNA synthesis than GD11 and camptothecin.

EXPRSESSION OF COLLAGEN TYPE I RESULTS EXPRSESSION OF COLLAGEN TYPE I Control Treatment of the cells with 25 μM of the compounds showed inhibitory effect of GD41 and GD9 (by 48.4% and 61.2%) but stimulatory effect of GD11 (by 30.8%) on type I collagen expression.

EXPRESSION OF INTEGRIN α2 RESULTS EXPRESSION OF INTEGRIN α2 Control Collagen type I synthesis is regulated by the signals mediated by activated 2β1-integrin receptors. Decrease in expression of α2β1 receptor was more pronounced by GD41 (90.9%) than GD9 (44.4%). In contrast, GD11 exerted stimulatory effect on type I collagen receptor (by 80%).

MMP-2 and MMP-9 activity Control GD41 GD9 GD11 proMMP-9 MMP-9 MMP-2 kD 120 100 85 70 60 50 40 proMMP-9 MMP-9 MMP-2 Control GD41 GD9 GD11 Activity of MMP degrading ECM components such as collagen was determined by zymography. Activity of both forms of MMP-9 (as proenzyme and active form) and MMP-2 were detected in polyacrylamide gels impregnated with gelatine.

MMP-2 and MMP-9 activity MMP-9 Densitometric analysis of the MMPs activity revealed that all new synthetic tylophorine analogs (GD9, GD11 and GD41) inhibited MMP-9 and MMP-2 activities, particularly compound GD11 which evoked stronger action in relation to MMP-9.

MMP-2 and MMP-9 activity MMP-2

CONCLUSION The results allowed to conclude that these compounds have a strong potential as a candidate anticancer drugs with different mechanism of their actions. The GD41 exhibited the highest cytotoxic and antiproliferative properties against MCF-7 The GD11 with stymulatory effect on collagen type I and its receptor and inhibitory effect on MMP-2 and MMP-9 comprises an important agent which requires further study.

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