Cancer Chemotherapy and Pharmacology (2019) 84:359–371 https://doi.org/10.1007/s00280-019-03863-w ORIGINAL ARTICLE Exposure time versus cytotoxicity for anticancer agents David M. Evans1 · Jianwen Fang3 · Thomas Silvers1 · Rene Delosh1 · Julie Laudeman1 · Chad Ogle1 · Russell Reinhart1 · Michael Selby1 · Lori Bowles1 · John Connelly1 · Erik Harris1 · Julia Krushkal3 · Larry Rubinstein3 · James H. Doroshow2 · Beverly A. Teicher2,4 Received: 11 February 2019 / Accepted: 2 May 2019 / Published online: 17 May 2019 © This is a U.S. government work and not under copyright protection in the U.S.; foreign copyright protection may apply 2019 Abstract Purpose Time is a critical factor in drug action. The duration of inhibition of the target or residence time of the drug molecule on the target often guides drug scheduling. Methods The efects of time on the concentration-dependent cytotoxicity of approved and investigational agents [300 com- pounds] were examined in the NCI60 cell line panel in 2D at 2, 3, 7 and in 3D 11 days. Results There was a moderate positive linear relationship between data from the 2-day NCI60 screen and the 3-, 7- and 11-day and a strong positive linear relationship between 3-, 7- and 11-day luminescence screen IC50s by Pearson correlation analysis. Cell growth inhibition by agents selective for a specifc cell cycle phase plateaued when susceptible cells were growth inhibited or killed. As time increased the depth of cell growth inhibition increased without change in the IC 50. DNA interactive agents had decreasing IC50s with increasing exposure time. Epigenetic agents required longer exposure times; several were only cytotoxic after 11 days’ exposure. For HDAC inhibitors, time had little or no efect on concentration response. There were potency diferences amongst the three BET bromodomain inhibitors tested, and an exposure duration efect. The PARP inhibitors, rucaparib, niraparib, and veliparib reached IC50s < 10 μM in some cell lines after 11 days. Conclusions The results suggest that variations in compound exposure time may refect either mechanism of action or com- pound chemical half-life. The activity of slow-acting compounds may optimally be assessed in spheroid models that can be monitored over prolonged incubation times. Keywords CxT · NCI60 · Concentration times time · Epigenetic agents Introduction Electronic supplementary material The online version of this Frequently, the cytotoxicity of anticancer drugs can be esti- article (https ://doi.org/10.1007/s0028 0-019-03863 -w) contains mated as the product of concentration and time for direct supplementary material, which is available to authorized users. cytotoxins such as DNA-damaging agents. However, for cell cycle-specifc agents, maximal cytotoxicity is dependent on * Beverly A. Teicher [email protected]; [email protected] cellular generation/doubling time [1, 2]. Since anticancer therapeutics have moved away from cytotoxic agents to epi- 1 Molecular Pharmacology Group, Leidos Biomedical genetic modifers and targeted drugs, the efect of time on Research, Inc., Frederick National Laboratory for Cancer cellular response needs to be re-examined. Cell cycle selec- Research, Frederick, MD 21702, USA tivity is an important factor in understanding the efect of 2 Developmental Therapeutics Program, Division of Cancer time on cytotoxicity. Drugs such as methotrexate and cyto- Treatment and Diagnosis, National Cancer Institute, Rockville, MD 20852, USA sine arabinoside are specifc for cells in S-phase; while drugs such as vincristine and paclitaxel are specifcally cytotoxic 3 Biometric Research Program, Division of Cancer Treatment and Diagnosis, National Cancer Institute, Rockville, toward cells in M-phase. Most drugs are more cytotoxic MD 20852, USA toward cells in exponential growth than toward plateau or 4 Molecular Pharmacology Branch, National Cancer Institute, stationary phase cells. RM 4-W602, MSC 9735, 9609 Medical Center Drive, Bethesda, MD 20892, USA Vol.:(0123456789)1 3 360 Cancer Chemotherapy and Pharmacology (2019) 84:359–371 Many approved drugs are relatively stable in cell culture at: https ://dtp.nci.nih.gov/branc hes/dscb/oncol ogy_drugs medium but have variable half-lives in human circulation et_expla natio n.html) and an investigational agent library, (Supplemental Table 1; [3, 4]). Doxorubicin, actinomycin acquired by synthesis or purchase. D, bleomycin, and vinblastine were stable in cell culture media for up to 10 days; however, these drugs have circulat- NCI60 screen ing half-lives in humans of 14.2, 14, 4, and 2.9 h, respec- tively. In cell culture media, etoposide lost 60% activity in The cell lines were grown in RPMI 1640 medium (Life 10 days; by contrast, the circulating half-life for etoposide Technologies, Gibco) containing 5% fetal bovine serum in humans is 3.62 h [5]. By HPLC, mitomycin C was stable (Life Technologies, HyClone, Logan, UT) and 2 mM for 7 days at 37 °C in cell culture media but doxorubicin L-glutamine. For experiments, cells were plated in 96-well had a half-life of 10–20 h under the same conditions [6]. In plates in 100 μl at cell numbers ranging from 5 × 103 to human circulation, mitomycin has a half-life of 0.81 h and 4 × 104 cells/well. The plates were incubated at 37 °C in doxorubicin of 14.2 h. Cisplatin had a half-life of 48 h in humid 5% CO2, for 24 h prior to compound addition. Com- cell culture media due to chemical reactivity and has a half- pounds in DMSO were added at fve concentrations from life of 0.44 h in human circulation [7, 8]. Decitabine, on the 100 to 0.01 μM. Following compound addition, the plates other hand, was relatively stable in cell culture media but has were incubated for 48 h at 37 °C, in humidifed incubators a plasma half-life of 20 min in humans’ due to high levels of with 5% CO 2. For adherent cells, the assay was terminated liver cytidine deaminase which metabolizes the drug to an by addition of cold TCA (fnal concentration, 10% TCA) inactive species [9]. The hydroxamic acid class of histone and incubated for 60 min at 4 °C. The plates were washed deacetylase inhibitors including vorinostat, belinostat, and with water and air-dried. Sulforhodamine B (SRB) solution panobinostat had very limited stability (1 day) in aqueous (100 μl) at 0.4% (w/v) in 1% acetic acid was added, and solution, were not stable in human plasma but had better plates were incubated for 10 min at room temperature. After stability in human serum (Supplemental Table 1; [10–12]). staining, unbound dye was removed by washing with 1% The mercaptoacetamide class of histone deacetylase inhibi- acetic acid and the plates were air dried. Bound stain was tors exemplifed by romidepsin was, in general, more stable solubilized with 10 mM trizma base, and the absorbance in aqueous solution, and plasma than hydroxamic acids [13]. read at 515 nm. For suspension cells, the methodology was The current study explores the efect of exposure time the same except that the assay was terminated by fxing set- (2–11 days) on the cytotoxicity and growth inhibition of tled cells at the bottom of the wells by gently adding 50 μl several classes of anticancer agents. of 80% TCA (fnal concentration, 16% TCA). 3‑ and 7‑day monolayer screen Material and methods 12 cell lines (11 lines and A549/ATCC) were screened per Cell lines run. Day 1, the cells were collected and suspended in 300 ml of media (RPMI 1640 supplemented with 5% FBS and NCI60 cell lines were obtained from the NCI Developmental 2 mM L-glutamine), then plated in 42 μl in 384-well plates Therapeutics Program Tumor Repository. For each lot of (CulturPlates, PE, Waltham, MA) using a Tecan Freedom cells, the Repository performed Applied Biosystems Amp- Evo. After incubation overnight, the Tecan Evo was used for FLSTR Identifler testing with PCR amplifcation to confrm compound addition. Each compound was added at 9 concen- consistency with the published. trations (1.5 nM–10 μM; DMSO concentration 0.25%), then Identifler STR profle for the given cell line [14, 15]. the plates were incubated for 3 or 7 days. The incubation was Each cell line was tested for mycoplasma when it was terminated by adding Cell Titer Glo (Promega, Madison, accepted into the repository; routine mycoplasma testing WI), and luminescence was determined. All assays were of lots was not performed. Cells were kept in continuous performed in triplicate. culture for no more than 20 passages. The optimal seed- ing densities for each of the cell lines for each time point 11‑day spheroid screen assessed were determined prior to performing the concentra- tion response studies [16]. All steps in the assay were performed using an automated high-throughput screening system consisting of two robotic Compounds arms interfaced with liquid handlers, pipettors, automated CO2 incubators, dispensers, shakers and plate readers. The Three hundred compounds were selected for the assays from screen was controlled via Momentum scheduling soft- the FDA-approved anticancer drugs (available from NCI ware (Thermo, Waltham, MA). Day 0: cells were plated in 1 3 Cancer Chemotherapy and Pharmacology (2019) 84:359–371 361 384-well Ultra Low Attachment (ULA) plates (Corning, Results NY) at densities ranging from 1 × 103 to 2 × 104 in RPMI media containing 10% fetal bovine serum in a volume of The NCI60 screen is the best known and most widely used 42 μL per well [16]. Spheroid formation and morphology compound cell-based screen to determine potential anti- were monitored using brightfeld imaging on a Cytation cancer activity [18–20]. The screen includes human tumor 3 high-content imaging system (BioTek, Winooski, VT) cell lines from nine major tumor types and uses a sulforho- equipped with a 4 × objective. Images were captured dig- damine B visible absorption endpoint.