In Vitro Characterization of Docetaxel as a Radiosensitizer in Canine and Feline Cancer Cell Lines

An in vitro study was conducted to investigate docetaxel as a radiation sensitizer in four canine (mammary carcinoma— CMT12 and CMT25, osteosarcoma—OS2.4, and transitional cell carcinoma—PTCC), and one feline cancer cell line (oral squamous cell carcinoma—SCCF1) to provide a basis for combination therapy in clinical patients. Cells were exposed to docetaxel followed by a single dose of radiation. The percent surviving fraction was determined by MTT assay. The combination index (CI) method determined synergistic cytotoxicity for the CMT12, CMT25 and OS2.4 cell lines with median CI values of 0.35, 0.47, 0.63 respectively. The SCCF1 cell line had moderate synergistic cytotoxicity with a median CI of 0.76, while the PTCC cell line resulted in antagonistic cytoxicity with a median CI of 2.75. The results indicated that docetaxel was a radiation sensitizer in 4 out of the 5 cancer cell lines tested.


Introduction
Chemoradiotherapy, with administration of chemotherapeutic agents prior to and during the course of radiation therapy, is increasingly being used in veterinary cancer therapy 1) as a radiation sensitizer, to improve the response to local radiation; 2) for the treatment of advanced locoregional disease; and/or 3) for both local as well as systemic effects on those tumors with a high metastatic potential [1][2][3][4][5][6][7].The clinical investigation of combination chemotherapy and radiation therapy in veterinary medicine has historically been limited to retrospective observational studies.A coordinated and scientific approach to the advancement of combination therapy in the clinic mandates preliminary in vitro studies of the combined cytotoxicity of chemotherapeutic agents and radiation.In order to realize improvements in response to radiation therapy it is preferable to combine it with a chemotherapeutic agent that has proven efficacy in the treatment of the tumor type under investigation.Additionally, the chemotherapeutic agent must have a potentiating effect or exhibit enhancement of the radiation response in the tumor that is greater than the enhancement of local normal tissue toxicity, thus achieving the goal of increasing the therapeutic index.It is also necessary to determine how best to combine radiation and chemotherapy, specifically in reference to scheduling and dosing of chemotherapy relative to the radiation therapy.
Docetaxel is a semisynthetic taxane whose primary mechanism of cytotoxicity is enhanced tubulin polymerization and inhibition of microtubule depolymerization [14].The initial rationale for using docetaxel as a radiation enhancer is that taxanes arrest proliferating cells in the G 2 M phase of the cell cycle which is the most radiosensitive cell cycle phase [15].The reason for this differential-phase sensitivity is that the endogenous radioprotectors, molecules containing sulfhydryl groups, are at their lowest concentrations in the cell during G 2 M. Sulfhydryl-group mediated cytoprotection results from both free-radical scavenging that protects against oxygen freeradical generation by ionizing radiation, and hydrogenatom donation to facilitate direct repair at sites of DNA damage.Docetaxel induces mitotic arrest and apoptosis in a concentration-dependent fashion and at low concentration can induce apoptosis without G 2 M arrest [16][17][18].Other mechanisms have been elucidated that may contribute to enhanced cytotoxicity in combination with radiation including: 1) the ability of docetaxel to eliminate radioresistant S-phase cells; 2) the ability of docetaxel to cause tumor reoxygenation; 3) the stimulation of antitumor immune resistance mechanisms; and 4) the inhibition of tumor angiogenesis at low docetaxel concentra-tions by inhibiting endothelial cell proliferation, migration and differentiation [19][20][21].An additional attribute of docetaxel is that it does not activate the orphan nuclear receptor SXR which is critical in the development of drug resistance, whereas paclitaxel has been shown to activate SXR and enhance ABCB-1 (MDR1, P-glycoprotein [P-gp]) mediated drug efflux [22].Docetaxel has been shown to have a radiosensitizing effect in vitro in human colon, lung, head and neck and cervical cancer cell lines [15,20,23,24].Furthermore, docetaxel has been shown to increase the therapeutic index when combined with radiation therapy in vivo in murine human xenograft cancer models as well as Phase I/II trials in humans with advanced cancer [25][26][27][28].
Alternative schedules of administration of docetaxel have been investigated that complement the use of docetaxel in combination with radiation therapy and are distinct from the scheduling of single agent docetaxel [29,30].Based on these in vitro schedules, docetaxel has proven effective in clinical trials involving humans with head and neck carcinoma treated with combination radiation therapy with weekly docetaxel and a Phase I/II docetaxel plus concurrent hyperfractionated radiotherapy in locally advanced unresectable head and neck cancer (TAX.ES1.102study) [27,28,[31][32][33].
Our long term goal is to define the role of docetaxel (DT) as a radiation sensitizer in the treatment of canine and feline cancer.Previously reported, phase I dose escalation studies in dogs and cats with spontaneous tumors determined the maximally tolerated dose of oral docetaxel [34,35].A phase II study to determine the efficacy of oral docetaxel in the management of canine epithelial tumors has been completed.In this study canine oral squamous cell carcinoma was found to be responsive to oral docetaxel [36].Furthermore a phase II study to determine the efficacy of oral docetaxel in the management of feline epithelial tumors targeting oral squamous cell carcinoma is currently ongoing.
The current series of in vitro experiments was performed to investigate the hypothesis that docetaxel would synergistically enhance the cytotoxicity of radiation therapy in canine and feline cancer cell lines in vitro.If this hypothesis was confirmed the studies would provide a basis for combination therapy of docetaxel plus radiation in the veterinary oncology clinic.In order to test this hypothesis we performed in vitro studies of the cytotoxicity of radiation therapy alone, docetaxel alone and then varying concentrations of docetaxel combined with a range of radiation exposures against different canine and feline cancer cell lines.

Canine and Feline Tumor Cell Lines
Canine mammary carcinoma cell lines (CMT12 and The canine mammary carcinoma cells were grown in Leibovitz L-15 media supplemented with 2.0 mM glutamine, 1 mM sodium pyruvate, 10 mM HEPES buffer, and 0.15% sodium bicarbonate solution (Invitrogen Inc.Carlsbad, CA, USA).The canine transitional cell carcinoma and osteosarcoma cell lines were grown in RPMI 1640 (Invitrogen Inc.Carlsbad, CA, USA).The feline SCC cells were grown in DMEM high glucose media (Dulbecco's modified eagle medium, Invitrogen Inc. Carlsbad, CA, USA).All media were supplemented with 10% fetal bovine serum and a 1% antibiotic/antimycotic solution (consisting of 100 U penicillin/100 ug streptomycin/0.25 µg amphotericin B per milliliter of media) (Invitrogen Inc.Carlsbad, CA, USA).All cell lines were maintained in a humidified atmosphere of 95% Air/5% CO 2 and incubated at 37˚C.Cells were seeded in triplicate at 2 -5 × 10 4 cells per well depending on the cell type into 96 well microtiter plates in a fixed volume of 100 µl of complete media and incubated overnight in a humidified atmosphere of 95% Air/5% CO 2 at 37˚C to allow cell attachment prior to experimental conditions.

Docetaxel
Docetaxel (Taxotere TM ), was obtained from Aventis Pharmaceuticals Products Inc., (Collegeville, PA) and prepared as described on the package insert.The initial docetaxel stock solution in ethanol was stored at -80˚C.Immediately prior to each experiment the docetaxel was thawed and diluted with culture medium to the final concentrations to be used in the cytotoxicity experiments.The final concentration of ethanol and polysorbate 80 in the culture media used in experiments never exceeded 0.1% and 0.0025% respectively.
Twenty-four hours after seeding into 96 well microtiter plates the complete medium was replaced with fresh serum-free medium alone or serum-free medium containing docetaxel at concentrations ranging from 0 -100 µM for 4 hours in a humidified atmosphere of 95% Air/5% CO 2 at 37˚C.Following incubation this medium was removed and the cells washed with HBSS (Hank's Balanced Salt Solution, Invitrogen Inc. Carlsbad, CA, USA) and fresh complete medium was added.Cells were either incubated without further treatment or irradiated as described below.Untreated controls and cells treated with radiation only were also sham-treated by rinsing with HBSS.

Radiation
Irradiation of the cell lines was performed using a standard procedure.A Siemens 6 MV linear accelerator was used to deliver radiation at a dose rate of 200 cGy/minute.Plated cells were irradiated under conditions of full dose buildup with 1.5 cm of tissue equivalent bolus material placed in the path of the radiation beam and with the gantry of the linear accelerator at 180˚.The cells were exposed to a single radiation dose which ranged from 0 to 10 Gy in 2 Gy increments after which they were incubated in a humidified atmosphere of 95% Air/5% CO 2 at 37˚C for 72 hours.

Cell Viability Assay
An MTT assay was performed 72 hours after experimental conditions described above.Briefly, the cells were incubated at 37˚C for one hour in 180 µl of serum-free RPMI 1640 media without phenol red and 20 µl of 5mg/ml MTT solution (Sigma-Aldrich, St Louis, Mo).The supernatants were removed and 200 µl of 2-propanol was added to each well to stop the MTT reaction.The cells were solubilized with repeat pipetting within the plate and absorbance readings were performed on a Tecan Safire microplate reader (Tecan, Raleigh, NC, USA) using a measurement wavelength of 540 nm and reference wavelength of 630 nm.

Analysis of the Combination Effects of Docetaxel with Radiation and Statistical Methods
The determination of cytotoxic activity of the combination of docetaxel and radiation was done using the combination index (CI) method of Chou-Talalay [37,38].The general equation for the classic isobologram is given by: where (D x ) 1 and (D x ) 2 in the denominators are the concentrations/doses for D 1 (docetaxel) and D 2 (radiation) alone that give x% inhibition, whereas (D) 1 and (D) 2 in the numerators are the concentrations/doses of docetaxel and radiation in combination that also inhibit x% (i.e., isoeffect).The (D x ) 1 or (D x ) 2 for docetaxel and radiation can be readily calculated from the median-effect equation of Chou [39]: where f a is the fraction affected and D m is the median effect concentration or dose (IC 50 or ID 50 ) that is obtained from the antilog of the X-intercept of the median effect plot, X-log (D) versus Y = log [f a /(1 -f a )] or D m = 10 −(y-intercet)/m , and m is the slope of the median effect plot.
For conservative mutually nonexclusive isobolograms of two agents, a third term is added.The third term is usually omitted, when the mutually exclusive (α = 0) assumption or classic isobologram is used [37,38]: The software program "Calcusyn" (Biosoft™, Cambridge, UK and USA) was used to perform these analyses and create a CI-isobologram for each combination.Graphical representations of the data from this analysis were generated with the CI on the y-axis and the fa (fraction of effect affected i.e. the CI for a particular % cell death e.g.0.3 for 30%) on the x-axis.A CI value between 0.9 and 1.1 indicates only additivity.Moderate synergism is depicted by CI values between 0.7 and 0.9 and clear synergism by CI values below 0.7.Antagonism is depicted by CI values above 1.1.

Cytotoxic Effects of Docetaxel, Radiation, and Docetaxel Combined with Radiation
Docetaxel was cytotoxic to all cell lines in a concentration dependent manner (Figure 1) with an EC 50 which ranged from 0.3 -5.3 µM, depending on the cancer cell line.All the cell lines were relatively resistant to the cytotoxic effects of a single radiation exposure of up to 10 Gy (Figure 2) with the ID 50 (5.7 Gy) only being defined for OS 2.4 cells.To determine whether the combination of docetaxel and radiation therapy resulted in synergistic cytotoxicity, the combination index was determined for each cell line.The combination of docetaxel and radiation resulted in synergistic cytoxicity for all cancer cell lines tested (Table 1 and Figures 3(a)-(d) for CI) with the exception of the transitional cell carcinoma cell line (PTCC) in which it showed antagonistic cytotoxicity, with a median CI of 2.75 (Figure 3(e)).

Discussion
Docetaxel's potential as a radiation sensitizer in veterinary patients with carcinomas/sarcomas was investigated in 4 animal carcinoma cell lines (3 canine, and 1 feline) and 1 canine osteosarcoma cell line.Cell viability was assessed using the MTT assay in control cells, cells exposed to docetaxel or radiation alone and cells exposed to docetaxel followed/combined with a single dose of radiation.Exposure to docetaxel alone resulted in concentration  Table 1.The median combination index for cells exposed to docetaxel and radiation.Synergism CI < 0.7; moderate synergism CI = 0.7 -0.9; Additive effect CI = 0.9 -1.1.dependent cytotoxic effect in all cell lines.In contrast, but not surprisingly, all cell lines studied were relatively resistant to the cytotoxic effects of single dose monotherapy up to the maximum 10 Gy radiation exposure studied.However, when these animal cancer cell lines were treated with the combination of docetaxel and radiation a synergistic cytotoxic effect was observed in canine mammary carcinoma (CMT-12 and CMT-25) cells and osteosarcoma cells (OS2.4).The studies on the feline squamous cell carcinoma cell line (SCCF1) revealed a moderate synergistic effect of the docetaxel-radiation cytotoxic effect was noted in the transitional cell carcinoma cell line (PTCC).
In order for drugs to combination, but somewhat surprisingly an antagonistic sensitize tumor cells to the cytoto rapy to be effective in the patient th (SCCF1) treated with doce xic effects of radiation increased cell kill needs to be either additive or synergistic using both modalities sequentially with a true clinical benefit only being achieved if the cytoxicity is greater to the tumor cells than to normal tissues.The observed additivity or synergism in these in vitro treatment models does not necessarily accurately predict the same results in vivo but provides a strong scientific rationale to perform a phase I clinical trial in veterinary patients with such cancers.Cellular radiosensitivity is dependent on the phase of the cell cycle cells are in, with cells in the S phase being most resistant to radiation damage and cells in the G 2 M phase of the cell cycle being most sensitive to radiation damage.Taxanes were shown in in vitro models to synchronize tumor cells in the G 2 M phase by forming high-affinity bonds with microtubules, promoting tubulin polymerization and stabilization thus facilitating greater sensitivity to the cytotoxic effects of radiation therapy.Additional mechanisms in vivo include the effects of tumor shrinkage which improves vascular perfusion and therefore oxygenation.As hypoxic regions of a tumor become better oxygenated they are more sensitive to the cytotoxic effects of radiation.Other postulated mechanisms of enhanced cytotoxicity when docetaxel and radiation are combined include inactivation of the anti-apoptotic protein Bcl-2 by phosphorylation and inhibition of angiogenesis [21,[40][41][42][43][44].
For chemoradiothe ere must be enhancement of the radiation response in the tumor that is greater than the enhancement of any local normal tissue toxicity thereby increasing the therapeutic index.In studies by Mason et al. [25] mice treated with intravenous docetaxel and radiation showed a potentiation of tumor radioresponse that was greater than the potentiation of jejunal crypt injury at all time points [25].In this same study when intravenous docetaxel was given to mice 48 hours prior to a single dose of radiation there appeared to be a radioprotective effect on the jejunum.Clinical studies involving tumor bearing dogs and cats would be necessary to determine if the same effect was seen in this population.
The feline SCC cell line taxel resulted in dose dependent cytotoxicity and an overall moderate synergistic cytotoxicity when combined with radiation.However, at low fractions affected (which relate to the lower docetaxel concentrations studied, 0.01 -0.1 µM) there was clearer evidence of synergistic cell kill when combined with radiation (CI = 0.62).Cats with oral SCC often present late in the disease with non-resectable tumors either due to advanced local disease or tumors in locations not amenable to surgical resection.T therapy has not improved survival times.Previous clinical studies evaluating gemcitabine as a radiation sensitizer in feline oral SCC revealed disappointing results with short disease free intervals or unexpected toxicity [45,46].Studies in humans with head and neck squamous cell carcinoma have shown improved outcomes with the addition of docetaxel to multimodality therapy [47,48].The results of the in vitro study presented here are en-diation sensitizer for feline oral squamous cell carcinoma is therefore warranted.
The results of our in vitro studies generate hypotheses for further in vitro studi docetaxel radiosensitization in canine and feline tumor cell lines.Importantly the data also provide a strong scientific rationale for the development and performance of early clinical trials to determine the optimal dosing and schedule of docetaxel combined with radiation in tumor bearing dogs (with e.g.mammary carcinoma or osteosarcoma) and cats with oral squamous cell carcinoma.

Conflict of Interest Statement
None of the authors of this paper has a fi sonal relationship with other people or orga nancial or pernizations that nowledgements Cornell University Dean's , and the Sprecher Institute
w, "Treatment with could inappropriately influence or bias the content of the paper.

Ack
This CMT25) were provided by Allison Church Bird, Auburn University College of Veterinary Medicine, Auburn, AL 36849.The canine osteosarcoma cell line (OS2.4)was provided by Dr. Katrina Mealey, Washington State University College of Veterinary Medicine, Pullman, WA 99164.The canine transitional cell carcinoma cell line (PTCC) was provided by Dr. Deborah Knapp, Purdue University College of Veterinary Medicine, WestLafayette, IN 47907.The feline oral squamous cell carcinoma (SCC) cell line (SCCF1) was provided by Dr. Thomas Rosol, The Ohio State University College of Veterinary Medicine, Columbus, OH 43210.