Endothelial toxicity profiling of anti-cancer ... · Endothelial toxicity profiling of anti-cancer...
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Jagiellonian Centre for Experimental Therapeutics ACKNOWLEDGMENTS: This study was supported by European Union from the resources of the European Regional Development Fund under the Innovative Economy Programme (grant coordinated by JCET-UJ, No POIG.01.01.02-00-069/09), as well as the Polish Ministry of Science and Higher Education (grant No. N N401 039637). Authors thanks Krzysztof Brzózka (Selvita SA) for reference kinases inhibitors. Endothelial toxicity profiling of anti-cancer chemotherapeutics 1 1 1,2 3 4 Tomasz Wojcik , Ewa Szczesny , Katarzyna Majzner , Justyna Miszczyk , Malgorzata Lukawska , 4 1,2 3 1,5 Ireana Oszczapowicz , Malgorzata Baranska , Wojciech Kwiatek , Stefan Chlopicki References: [1] Laverty HG et al. (2011) How can we improve our understanding of cardiovascular safety liabilities to develop safer medicines? Br J Pharmacol 163(4); 675-693 [2] Soultati A et al. (2012) Endothelial vascular toxicity from chemotherapeutics agents: preclinical evidence and clinical implications. Cancer Treat Rev 38; 473-483 [3] Al D et al. (2013) Chemotherapy induced cardiovascular toxicity: Beyond anthracyclines. Minerva Anestesiol [Epub ahead of print] 1 2 Jagiellonian Centre for Experimental Therapeutics (JCET), Bobrzynskiego 14, Krakow, Poland; Faculty of Chemistry, Jagiellonian University, Ingardena 3, 30-060 Krakow, Poland; 3 4 Department of Experimental Physics of Complex Systems, The H. Niewodniczański Institute of Nuclear Physics Polish Academy of Sciences, Krakow, Poland; Institute of Biotechnology and Antibiotics, 5 5 Staroscinska, 02-516 Warsaw, Poland; Department of Experimental Pharmacology, Chair of Pharmacology, Jagiellonian University, Medical College, Grzegorzecka 16, Krakow, Poland Introduction Cardiovascular toxicity is major reason for drug attrition during pre- clinical phases of drug development [1] and could be due to vascular damage, induced by a direct endothelial damage, activation of coagulation factors or vascular inflammation. Since endothelial dysfunction constitutes an integrative response predictive for future cardiovascular events [2] we aimed to develop a comprehensive panel of endothelial cytotoxicity screening assays based on High-content screening (HCS) automated fluorescence microscopy, Raman microscpectroscopy, chemiluminescence and biochemical analysis. Using this approach we screened a group of classical and targeted chemotherapeutics. Genotoxicity - cytokinesis blocked micronucleus (CBMN) assay Effects of anthracyclines on nuclear area (chirarchical clustering) Intranuclear accumulation of anthracyclines (Raman microspectroscopy) Apoptosis - caspases 3/7 activities (chemiluminescence) Conclusions We demonstrated that anthracyclines (e.g. Doxorubicin, Daunorubicin) accumulate in the nucleus, causing nuclei swelling, genotoxicity as well as pro-inflammatory endothelium phenotype in nanomolar concentrations. Anthracycline epimers were less toxic (e.g. Epidoxorubicin, Epidaunorubicin). Nonspecific cycline-dependent kinase inhibitors (Dinaciclib, Flavopiridol), displayed high endothelial cytotoxicity linked to a decrease in mitochondrial membrane potential and oxidative stress. In turn, endothelial toxicity of tyrosine kinases inhibitors was featured by gross phospholipidosis in particular for Sunitinib and Imatinib in concentrations near to their therapeutic Cmax. In conclusion, multi-parameter HCS in human endothelium could assist in lead compound selection in early drug development and the development of safer drugs, with decreased vascular toxicity and lower risk of cardiovascular adverse effects. contact to: [email protected] Reactive oxygen species generation (Dihydroethidium) Fosfolipidosis - neutral lipids (LipidTOX) The results presented shows cell cycle delay in endothelial cells exposed to nanomolar concentrations of drugs. Decrease in NDI in cells treated with daunorubicin was markedly more prominent than in cells treated with doxorubicin . Both of drugs were found to induced DNA damage at nanomolar concentrations of compounds. The frequency of micronuclei was increased almost two times by daunorubicin at 100 nM concentration, when compare to doxorubicin at the same concentration (107 vs. 50). To examine whether changes in the nuclear area of endothelium may be directly linked to DOX and DNR accumulation in the nucleus, two-dimensional spectral maps and chemical images, calculated by integration the intensity of the anthracycline fluorescence maximum range, were acquired by Raman microspectroscopy. Integrated Raman spectra at intensities in -1 -1 the region from 3820 cm to 4245 cm , representing DNR and DOX, were recorded to visualize the content and distribution of these compounds in endothelial cells. Nuclear area of endothelial cells stained with Hoechst 33342 seems to be simple and accurate of nuclear accumulation of anthracyclines. In order to test if epimers of doxorubicin (EDOX) and daunorubicn (EDNR) accumulates in lower amounts than doxorubicin (DOX) and daunorubicin (DNR), cells were treated with these drugs for 24 hours, docetaxel (DOCE) and 5-fluorouracil (5-FU) were used as negative controls in this assay. Obtained results were analysed using chierarchical clustering module of Spotfire software (Perkin Elmer). Anthracyclines have been for several decades the most studied agents because of their known cardiovascular effects and relatively high incidence of heart failure. Hovewer, cancer patients are currently treated with newer chemotherapeutics such as tyrosine kinases inhibitors or cycline-dependent kinases inhibitors that are also responsible of causing cardiovascular toxicities. The type of cardiovascular toxicity associated with these newer agents (type II) appers to be different that caused by anthracylines (type I) [3]. Thus, we examine the effects of these newer agents on endothalial cells, using HCS fluorescence microscopy. Endothelial toxicity of anthracyclines and kinases inhibitors DOX EDOX DNR EDNR EC [mM] 50 2,593 7,857 1,957 3,394 SD [mM] 0,491 0,049 0,885 0,273
Transcript of Endothelial toxicity profiling of anti-cancer ... · Endothelial toxicity profiling of anti-cancer...
Jagiellonian Centre for Experimental Therapeutics
ACKNOWLEDGMENTS: This study was supported by European Union from the resources of the European Regional Development Fund under the Innovative Economy Programme (grant coordinated by JCET-UJ, No POIG.01.01.02-00-069/09), as well as the Polish Ministry of Science and Higher Education (grant No. N N401 039637). Authors thanks Krzysztof Brzózka (Selvita SA) for reference kinases inhibitors.
Endothelial toxicity profiling of anti-cancer chemotherapeutics
1 1 1,2 3 4Tomasz Wojcik , Ewa Szczesny , Katarzyna Majzner , Justyna Miszczyk , Malgorzata Lukawska , 4 1,2 3 1,5Ireana Oszczapowicz , Malgorzata Baranska , Wojciech Kwiatek , Stefan Chlopicki
References:[1] Laverty HG et al. (2011) How can we improve our understanding of cardiovascular safety liabilities to develop safer medicines? Br J Pharmacol 163(4); 675-693 [2] Soultati A et al. (2012) Endothelial vascular toxicity from chemotherapeutics agents: preclinical evidence and clinical implications. Cancer Treat Rev 38; 473-483[3] Al D et al. (2013) Chemotherapy induced cardiovascular toxicity: Beyond anthracyclines. Minerva Anestesiol [Epub ahead of print]
1 2Jagiellonian Centre for Experimental Therapeutics (JCET), Bobrzynskiego 14, Krakow, Poland; Faculty of Chemistry, Jagiellonian University, Ingardena 3, 30-060 Krakow, Poland;3 4Department of Experimental Physics of Complex Systems, The H. Niewodniczański Institute of Nuclear Physics Polish Academy of Sciences, Krakow, Poland; Institute of Biotechnology and Antibiotics,
5 5 Staroscinska, 02-516 Warsaw, Poland; Department of Experimental Pharmacology, Chair of Pharmacology, Jagiellonian University, Medical College, Grzegorzecka 16, Krakow, Poland
IntroductionCardiovascular toxicity is major reason for drug attrition during pre-clinical phases of drug development [1] and could be due to vascular damage, induced by a direct endothelial damage, activation of coagulation factors or vascular inflammation. Since endothelial dysfunction constitutes an integrative response predictive for future cardiovascular events [2] we aimed to develop a comprehensive panel of endothelial cytotoxicity screening assays based on High-content screening (HCS) automated fluorescence microscopy, Raman microscpectroscopy, chemiluminescence and biochemical analysis. Using this approach we screened a group of classical and targeted chemotherapeutics.
Genotoxicity - cytokinesis blocked micronucleus (CBMN) assay
Effects of anthracyclines on nuclear area (chirarchical clustering)
Intranuclear accumulation of anthracyclines (Raman microspectroscopy)
Apoptosis - caspases 3/7 activities (chemiluminescence)
ConclusionsWe demonstrated that anthracyclines (e.g. Doxorubicin, Daunorubicin) accumulate in the nucleus, causing nuclei swelling, genotoxicity as well as pro-inflammatory endothelium phenotype in nanomolar concentrations. Anthracycline epimers were less toxic (e.g. Epidoxorubicin, Epidaunorubicin). Nonspecific cycline-dependent kinase inhibitors (Dinaciclib, Flavopiridol), displayed high endothelial cytotoxicity linked to a decrease in mitochondrial membrane potential and oxidative stress. In turn, endothelial toxicity of tyrosine kinases inhibitors was featured by gross phospholipidosis in particular for Sunitinib and Imatinib in concentrations near to their therapeutic Cmax. In conclusion, multi-parameter HCS in human endothelium could assist in lead compound selection in early drug development and the development of safer drugs, with decreased vascular toxicity and lower risk of cardiovascular adverse effects.
contact to: [email protected]
Reactive oxygen species generation (Dihydroethidium)Fosfolipidosis - neutral lipids (LipidTOX)
The results presented shows cell cycle delay in endothelial cells exposed to nanomolar concentrations of drugs. Decrease in NDI in cells treated with daunorubicin was markedly more prominent than in cells treated with doxorubicin . Both of drugs were found to induced DNA damage at nanomolar concentrations of compounds. The frequency of micronuclei was increased almost two times by daunorubicin at 100 nM concentration, when compare to doxorubicin at the same concentration (107 vs. 50).
To examine whether changes in the nuclear area of endothelium may be directly linked to DOX and DNR accumulation in the nucleus, two-dimensional spectral maps and chemical images, calculated by integration the intensity of the anthracycline fluorescence maximum range, were acquired by Raman microspectroscopy. Integrated Raman spectra at intensities in
-1 -1the region from 3820 cm to 4245 cm , representing DNR and DOX, were recorded to visualize the content and distribution of these compounds in endothelial cells.
Nuclear area of endothelial cells stained with Hoechst 33342 seems to be simple and accurate of nuclear accumulation of anthracyclines. In order to test if epimers of doxorubicin (EDOX) and daunorubicn (EDNR) accumulates in lower amounts than doxorubicin (DOX) and daunorubicin (DNR), cells were treated with these drugs for 24 hours, docetaxel (DOCE) and 5-fluorouracil (5-FU) were used as negative controls in this assay. Obtained results were analysed using chierarchical clustering module of Spotfire software (Perkin Elmer).
Anthracyclines have been for several decades the most studied agents because of their known cardiovascular effects and relatively high incidence of heart failure. Hovewer, cancer patients are currently treated with newer chemotherapeutics such as tyrosine kinases inhibitors or cycline-dependent kinases inhibitors that are also responsible of causing cardiovascular toxicities. The type of cardiovascular toxicity associated with these newer agents (type II) appers to be different that caused by anthracylines (type I) [3]. Thus, we examine the effects of these newer agents on endothalial cells, using HCS fluorescence microscopy.
Endothelial toxicity of anthracyclines and kinases inhibitors
DOX EDOX DNR EDNR
EC [mM]50 2,593 7,857 1,957 3,394
SD [mM] 0,491 0,049 0,885 0,273
