Synergy of Anti-Cancer Drugs to Induce Cell Death in ...
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APPLICATION NOTE Synergy of Anti-Cancer Drugs to Induce Cell Death in Pancreatic Cancer Cells Summary BRD4770 is a novel S-adenosylmethionine mimetic inhibitor of histone methyl transferase (HMT) G9a that promotes senescence in PANC-1 pancreatic cancer cells (which lack functional p53 and p16). Biolog PMM MicroPlates™ containing established anti-cancer agents were seeded with PANC-1 cells in pairwise combination with BRD4770 at different concentrations. Of four compounds which enhanced BRD4770- induced PANC-1 cell death, gossypol was found to be effective in acting synergistically to induce autophagy and cell death (1). Results Cellular metabolism and susceptibility to toxic chemicals was measured as previously described (2) using Biolog Phenotype MicroArray plates precoated with 92 different anti-cancer compounds, each at four different titrated concentrations. An example of one such plate (PM-M13) is depicted in Figure 1. For the assay, PANC-1 and hHPNE cells were resuspended in an RPMI-1640 based medium containing BRD4770 at 3 different concentrations and DMSO only (DMSO, 1.25uM, 2.5uM, and 5.0uM BRD4770), and seeded at 30,000 cells per well. After 48 hr incubation, Biolog Redox Dye Mix MB was added to each well, and cells were incubated in an OmniLog ® 50-plate incubator/reader for automated measurement of the kinetics of cell metabolism over 24 hrs. Four compounds enhanced cell death, as measured by decreased metabolic dye reduction (Figure 2). None of these “hit” compounds enhanced cell death in hHPNE, which expresses relatively low levels of HMT G9a. Additional testing showed that gossypol (wells D9-12) was most effective in synergizing with BRD4770 to kill PANC-1 cells. The natural product and putative BH3 mimetic, gossypol, enhanced the cytotoxicity of BRD4770 in a synergistic manner in p53-mutant PANC-1 cells but not in immortalized nontumorigenic pancreatic cells. Other data further suggested that these compounds act together to induce autophagy-related cell death in pancreatic cancer cells. Conclusions The Biolog Phenotype MicroArray system provides a simple format to enable discovery of potential synergistic killing of cancer cells by combining two effective anticancer agents. In this study, a novel small molecule inhibitor with activity against pancreatic cells was found to synergize with gossypol (Biolog PM-M13 MicroPlate). This unexpected synergy suggests new lines of mode-of-killing experiments and perhaps also novel approaches to treatment of pancreatic cancer. Figure 2: Compounds that enhance BRD4770-induced pancreatic cancer cell death identified by measuring live-cell metabolism. PANC-1 cells were seeded in 96-well plates that were pre-coated with cytotoxic drugs in four doses. BRD4770 was added to a set of plates while DMSO was added to another set of plates as negative controls for 48hr. The cellular metabolism levels were monitored by redox dye reduction in an OmniLog instrument for 24 hr. Four compounds enhanced BRD4770 induced pancreatic cell death in a dose-dependent manner. Figure 1: Biolog PM-M13 MicroPlate containing anti-cancer agents at 4 titrated concentrations ¹From: Y Yuan, AJ Tang, AB Castoreno, S-Y Kuo, Q Wang, P Kuballa, R Xavier, AF Shamj, SL Schreiber and BK Wagner, Cell Death and Disease (2013) 4, e690; doi;10.1038/cddiss.2013.191 ² From: Bochner BR, Siri M, Huang RH, Noble S, Lei X-H, Clemons, P.A., Wagner, B.K., (2011) Assay of the Multiple Energy-Producing Pathways of Mammalian Cells. PLoS ONE 6(3):e18147. doi:10.1371/journal.pone.0018147 00A 052 Rev A 1/2015
Transcript of Synergy of Anti-Cancer Drugs to Induce Cell Death in ...
A P P L I C A T I O N N O T E
Synergy of Anti-Cancer Drugs to Induce Cell Death in Pancreatic Cancer Cells
Summary
BRD4770 is a novel S-adenosylmethionine mimetic inhibitorof histone methyl transferase (HMT) G9a that promotessenescence in PANC-1 pancreatic cancer cells (which lackfunctional p53 and p16). Biolog PMM MicroPlates™ containingestablished anti-cancer agents were seeded with PANC-1 cellsin pairwise combination with BRD4770 at differentconcentrations. Of four compounds which enhanced BRD4770-induced PANC-1 cell death, gossypol was found to be effectivein acting synergistically to induce autophagy and cell death (1).
Results
Cellular metabolism and susceptibility to toxic chemicals wasmeasured as previously described (2) using Biolog PhenotypeMicroArray plates precoated with 92 different anti-cancercompounds, each at four different titrated concentrations. Anexample of one such plate (PM-M13) is depicted in Figure 1.For the assay, PANC-1 and hHPNE cells were resuspended inan RPMI-1640 based medium containing BRD4770 at 3different concentrations and DMSO only (DMSO, 1.25uM,2.5uM, and 5.0uM BRD4770), and seeded at 30,000 cells perwell. After 48 hr incubation, Biolog Redox Dye Mix MB wasadded to each well, and cells were incubated in an OmniLog®
50-plate incubator/reader for automated measurement of thekinetics of cell metabolism over 24 hrs.
Four compounds enhanced cell death, as measured bydecreased metabolic dye reduction (Figure 2). None of these “hit”compounds enhanced cell death in hHPNE, which expressesrelatively low levels of HMT G9a. Additional testing showed thatgossypol (wells D9-12) was most effective in synergizing withBRD4770 to kill PANC-1 cells. The natural product and putativeBH3 mimetic, gossypol, enhanced the cytotoxicity of BRD4770in a synergistic manner in p53-mutant PANC-1 cells but not inimmortalized nontumorigenic pancreatic cells. Other data furthersuggested that these compounds act together to induceautophagy-related cell death in pancreatic cancer cells.
Conclusions
The Biolog Phenotype MicroArray system provides a simpleformat to enable discovery of potential synergistic killing ofcancer cells by combining two effective anticancer agents. Inthis study, a novel small molecule inhibitor with activity againstpancreatic cells was found to synergize with gossypol (BiologPM-M13 MicroPlate). This unexpected synergy suggests newlines of mode-of-killing experiments and perhaps also novelapproaches to treatment of pancreatic cancer.
Figure 2: Compounds that enhance BRD4770-induced pancreaticcancer cell death identified by measuring live-cell metabolism.PANC-1 cells were seeded in 96-well plates that were pre-coated withcytotoxic drugs in four doses. BRD4770 was added to a set of plates whileDMSO was added to another set of plates as negative controls for 48hr. The cellular metabolism levels were monitored by redox dye reduction in anOmniLog instrument for 24 hr. Four compounds enhanced BRD4770induced pancreatic cell death in a dose-dependent manner.
Figure 1: Biolog PM-M13 MicroPlate containing anti-cancer agentsat 4 titrated concentrations
¹From: Y Yuan, AJ Tang, AB Castoreno, S-Y Kuo, Q Wang, P Kuballa, R Xavier, AFShamj, SL Schreiber and BK Wagner, Cell Death and Disease (2013) 4, e690;doi;10.1038/cddiss.2013.191
² From: Bochner BR, Siri M, Huang RH, Noble S, Lei X-H, Clemons, P.A., Wagner,B.K., (2011) Assay of the Multiple Energy-Producing Pathways of Mammalian Cells.PLoS ONE 6(3):e18147. doi:10.1371/journal.pone.0018147
00A 052 Rev A 1/2015
