Edited by: Kwan-Hwa Chi, Shin-Kong Memorial Hospital, Taiwan
Reviewed by: Jang-Yang Chang, National Health Research Institutes, Taiwan; Hsin-Ell Wang, National Yang-Ming University, Taiwan
This article was submitted to Radiation Oncology, a section of the journal Frontiers in Oncology.
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Despite objective response rates of 60–80% to platinum-based chemotherapy integrated with maximum cytoreductive surgery (
Assembly of chromosomes during the G2/M-phase of the cell cycle leaves cells not only vulnerable to death-provoking DNA double-strand breaks from ionizing radiation (
Clinically, women having ovarian cancers that relapse after platinum and paclitaxel-based chemotherapies have therapeutic responses to an 8 Gy × 3 fraction stereotactic ablative radiosurgery (SABR) (
Human ovarian cancer cells OVCAR3 [P-glycoprotein multiple drug resistance transporter 1 (
Radiation was delivered using a 137Cs γ-irradiator (JL Shepherd Associates, San Fernando, CA, USA) at 325 cGy per minute. Cabazitaxel (Jevtana, XRP6258) was an investigational agent provided to Case Western Reserve University (Cleveland, OH, USA) under an agreement with Sanofi-Aventis (Bridgewater, NJ, USA). To interfere with mitotic spindle activity (
Triplicate replicates of 1 × 104 OVCAR3, SKOV3, and TOV-112D cells were incubated in 96-well plates for each indicated cabazitaxel or paclitaxel dose. Cells either underwent sham irradiation or a conventional clinical radiation dose (2 Gy) at the start of cabazitaxel or paclitaxel exposure. Six hours after the indicated treatment, exchanges for drug-free medium were done. After 18 h (i.e., 24 h after the start of drug exposure), cells were re-incubated in 300 μL of drug-free medium plus MTT (3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide, 5 mg/mL). Following 3 h of incubation at 37°C, 96-well plates were analyzed by a spectrophotometer (Perkin Elmer, Waltham, MA, USA) for quantified colorimetric absorbance at an excitation wavelength of 540 nm. Means and standard errors (SE) reflecting viable cell number were plotted graphically. Analyses of fitted dose-response curves determined the amount of drug (IC50) that reduced viable cell number by 50% of control cells (
Exponentially growing OVCAR3, SKOV3, and TOV-112D cells were plated in triplicate on 24-well dishes to yield 300 cells (0, 2, 4, 8 Gy) or 3000 cells (20 Gy) per well. Cells underwent radiation or radiation with 6 h cabazitaxel (1 μM), an end concentration guided by human pharmacokinetic data (
Exponentially growing TOV-112D and SKOV3 cells were plated on 100 mm dishes to yield 2.0 × 106 colonies per dish. To study cell cycle-related molecular events at the G2/M-phase transition, cells underwent radiation (0 or 2 Gy) and/or 6 h 1 μM cabazitaxel treatment. Trypsinized cultures were fixed and stained for DNA content by propidium iodide (PI) for standard flow cytometry or 4,6-diamidino-2-phenylindole (DAPI) with fluorescent antibodies reactive with cyclin A2 and phospho-S10-histone H3 (PHH3) as described (
Ovarian cancer cell viability was determined by MTT assay. Figure
Figure
Cell survival after radiation or after radiation-cabazitaxel was established by clonogenic assays (Figure
Our next step explored the effects of 1 μM cabazitaxel on the proportion of cells residing in G2- and M-phases of the cell cycle by cytometry. To mimic clinical pharmacokinetics of cabazitaxel, drug-containing media was exchanged for drug-free media at the 6 h time point after initial cabazitaxel exposure. We focused cytometry upon the initial 24 h post-exposure cell cycle kinetics period because were interested in testing putative, clinical workday feasible radiation-cabazitaxel schedules. Table
Cell line | Hours after cabazitaxel |
||||
---|---|---|---|---|---|
0 h | 6 h | 12 h | 18 h | 24 h | |
OVCAR3 | 12 | 26 | 37 | 35 | 43 |
SKOV3 | 13 | 38 | 51 | 50 | 61 |
TOV-112D | 8 | 33 | 47 | 46 | 58 |
Then, four schedules of combined radiation-cabazitaxel treatment were investigated by cytometric assay and by clonogenic assay (Figure
The first schedule of 1 μM cabazitaxel given 24 h before radiation (24 h+) was supraadditive. Cell cycle analyses indicated that many cells had become stalled at the G2/M transition 24 h after cabazitaxel exposure (Figure
The second schedule of cabazitaxel 1 μM given 18 h before radiation (18 h+) was also supraadditive. Cytometry indicated that 18 h after cabazitaxel a lower cell proportion had stopped cell cycle progression at the G2/M transition (Figure
The fourth schedule of radiation followed 24 h later by a 6-h exposure to cabazitaxel 1 μM (24 h−) resulted in mixed subadditive or additive interactions. For this schedule, cytometry detected only minimal perturbations in cell cycle proportions at the start of cabazitaxel exposure (Figure
Drug-induced biologic effects as a function of the cell cycle may be monitored by DNA content flow cytometry and readily distinguish cell proportions residing in the G1 and S phases of the cell cycle. But cells with four genome complements (4C cells) can exist in G2, M, or 4C G1 phases that results from either endoreduplication or failure to undergo cytokinesis, giving rise to a bi-nucleate cell cycle. To explore the effects of cabazitaxel at the G2/M transition, we employed multi-parameter analysis using immunodetection of DNA content, cyclin A2 as an indicator of early mitosis, and phospho-S10-histone H3 whose elevated expression during the decline of cyclin A2 maps late mitosis.
Figure
Radiation (2 Gy) when added to 1 μM cabazitaxel treatment did not change the cabazitaxel effects reflected by cyclin A2 or PHH3 in SKOV3 cells. However, the presence of sub G1 events indicating fragmented DNA (and lethal cell events) appears enhanced in the irradiated SKOV3 cells. By comparison, irradiated TOV-112D cells showed 4C G2 arrest at 24, 48, and 72 h, a profound 4C G1 arrest at 48 h and at 72 h, and a mitotic arrest at 24 and 48 h that is linked to lower PHH3 levels. The latter finding indicates that the two cell lines are responding differently at the molecular level, and further partitioning of the G2/M-phase could detail such a molecular “fingerprint.” Taken together, high-content flow cytometry suggests cabazitaxel administered with or without radiation results in mitotic arrest followed by an escape to a higher order DNA content cell cycle that may or may not be further damaged by radiation.
A typical mitotic phenotype for microtubule poisons is arrest at metaphase (cyclin A2 levels are background) or prometaphase (chromosomes are not attached to a spindle). An expected cabazitaxel-induced phenotype is residence of cells in a PHH3 high/cyclin A2 low level state (M-LM state). Figure
Cell cycle phase impact upon cell radiosensitivity has been studied extensively (
A precise mechanism by which an augmented G2/M-phase cell cycle arrest sensitizes cells to radiation is not well understood. An enhanced susceptibility to radiation-induced double-strand breaks in tightly packaged DNA could partially explain the radiosensitivity we observed; however, such an explanation cannot account for the cytotoxic effects of cabazitaxel alone where mitotic checkpoint responses are likely to be active and protracted response activation may be lethal as well. Attempted G2/M to G1 traversal in the setting of unrepaired DNA damage is a toxic event, likely due to activation of mitotic cell death responses and possible loss of vital genetic material in cell progeny through unrepaired DNA double-strand breaks (
We put forward that stalling of G2/M-phase traversal by cabazitaxel explains supraadditive interactions when cabazitaxel is administered 18–24 h before ionizing radiation. This arrest mechanism is additionally supported by the resolution of the G2/M-phase block through either a mitotic spindle checkpoint response or endoreduplication. Administering radiation and cabazitaxel treatment together appears additive, as the cell cycle perturbing effects of cabazitaxel are not apparent for several hours later. Giving radiation and chasing it 24 h later with cabazitaxel results in mixed response. The cumulative data suggests that in a radiation-cabazitaxel therapeutic strategy, the optimal sequence is cabazitaxel first and radiation preferably 24 h later. Pharmacokinetic and pharmacodynamic experiments in mice have been published to guide human clinical trials (
Paclitaxel is utilized commonly in the clinical management of women with ovarian cancer (
Sanofi-Aventis provided a research grant to the Department of Radiation Oncology and The Case Western Reserve University School of Medicine whose funds supported some research activities related to this manuscript.
Funding: Case Comprehensive Cancer Center and Sanofi-Aventis.