Synergism in Concomitant Chemoradiotherapy of Cisplatin and Oxaliplatin and their Liposomal Formulation in the Human Colorectal Cancer HCT116 Model

Materials and Methods

Cell lines and drugs. The HCT116 colorectal carcinoma cell line obtained from American Type Culture Collection is p53-wild type. Cells were routinely cultured in modified Eagle's medium (MEM) (Sigma, Oakville, Canada) supplemented with 10% fetal bovine serum (FBS), 2 mM glutamine, 1 mM Sodium-Pyruvate, 100 units/ml penicillin and 100 μM streptomycin in a fully humidified incubator at 37°C in an atmosphere containing 5% CO₂. Cisplatin and oxaliplatin were purchased from Sigma-Aldrich. Lipoplatin™ and Lipoxal™, the liposomal formulation of cisplatin and oxaliplatin, respectively, were generously provided from Regulon Inc. (Athens, Greece). All drugs were diluted to the given concentrations in culture medium immediately before use in FBS-free MEM.

Clonogenic assay. The cytotoxicity of platinum and ionizing radiation against HCT116 cell line was assessed by a colony formation assay. For chemoradiation combination therapeutic studies on HCT116 cells, we performed with three groups of treatment: 1) the control group treated with four representative platinum-based drugs only at five concentrations, 2) the group treated with ionizing radiation at four doses of radiation, 3) the group treated with a combination of drugs and radiation, at a constant of the IC₅₀ and LD₅₀ values (IC₅₀ referred to, and LD₅₀ referred to the dose of radiation causing a 50% reduction of cell growth compared with untreated cells). All the experiments began by growing the 1000 cells from a single-cell suspension in 100-mm cell culture dishes containing 10 ml of culture medium and incubated at 37°C in a humidified atmosphere containing 5% CO₂ for 24 h. After the culture medium was removed, the cells were washed twice with PBS before following treatments.

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Figure 1.

Dose-response curve for radiotherapy-alone on HCT116 cells. Cells were irradiated with gamma-rays (γ-rays) during exponential cell growth using a ⁶⁰Co unit at a dose rate of 1.64 Gy/min. Each point represents the mean of 3 independent experiments and the bars indicate the standard deviation (S.D).

For chemotherapy-alone, cells were treated with platinum compounds at appropriate concentrations and further incubated for the desired period. Thereafter, the drugs were removed and the cells were incubated for another 7 days to allow the formation of colonies. Cells were fixed and stained with 0.1% crystal violet, after washing with tap water. Colonies containing more than 50 cells were counted manually to calculate the clonogenic survival fractions. Each experiment was performed at least of 3 times using triplicate cultures for each drug concentration. In addition, the cytotoxicity of platinum drugs alone was examined in order to determine the IC₅₀ values, i.e. the concentrations of drugs causing a 50% reduction of cell growth compared with untreated cells.

For radiation treatment, cells were irradiated with gamma-rays (γ-rays) during exponential cell growth using a ⁶⁰Co unit at a dose rate of 1.64 Gy/min. Cells were maintained in MEM supplemented with 10% FBS during all the radiation exposures, which were performed at room temperature. Dose response curves were established for the HCT116 cells using a total with 4 single doses of 1, 2, 3 and 5 Gy.

For concomitant chemoradiotherapy, cells were treated with the platinum compound at appropriate concentrations, which were the same concentrations as the ones used to study the cytotoxicity of chemotherapy-alone. Thereafter, the drug was removed and the cells were further incubated for the desired periods. We selected exposure time of 8 and 48 h as they were previously shown to correlate with the highest and lowest levels of DNA-platinum adducts, concentration in the nucleus, respectively (12). Then, the cells were irradiated with γ-rays at the LD₅₀ value (i.e., at a single dose of 2.3 Gy, obtained from the results of Figure 1). This figure shows the cell survival fraction decreased, as a function of radiation dose. The 2.3 Gy value was obtained on platinum-free HCT116 cells and corresponds to the radiation dose which resulted in a reduction of 50% of the colonies formation.

After treatment with either radiation-alone or concomitant chemoradiotherapy, the cells were incubated for an additional 7 days (to allow the formation of colonies) before cells fixation and counting colonies formation were performed as described above. Each experiment was performed at least 3 times using triplicate cultures for each drug concentration.

The result of colonies formation assays from different group (chemotherapy-alone, radiation-alone and combined treatments) were used to calculate the combination index (CI) in order to assess the levels of synergy, as described in the following section.

Determination of combination index: Antagonism/Synergism/Additivity. Evaluation of the nature of activity of platinum complexes in combination with radiation is mutually non-exclusive modalities, since the plots for drug and radiation and their combination are not parallel to each other. This evaluation and the CI calculation with respect to cell survival were performed according to the method described by Chou and Talalay (20). The CI was evaluated for each drug at 8 and 48 h of incubation. We also sought to determine whether the synergism would be reached at a platinum drug dose equivalent, lower and higher than that of respective IC₅₀. A lower concentration was defined as the average of minimum tested concentration to the concentration at IC₅₀ values, while a higher concentrations was defined as the average of concentration at IC₅₀ values to the maximum tested concentration.

Mathematical evaluation of the data was carried out by computerized analysis of the median dose effect (IC₅₀) and the combination index as well as linear correlation coefficient (r). According to the CALCUSYN software of BIOSOFT (Ferguson, MO, USA) developed by Chou (21), the CI is expressed by the relationship where each term represents, the denominator, (Ptx) is for the concentration of platinum compound “alone” that inhibits colonies formation at x%, and (IRx) is for the dose of radiation-alone that inhibits colonies formation at the same x%. In the numerator, (Pt) + (IR) “in combination” also inhibit the colonies formation also at the same x%. CI values <1.0, and =1.0, and >1.0 indicate synergistic, additive, and antagonistic effects, respectively.

Cell cycle and apoptosis analysis. Cells were treated with platinum drugs at concentrations corresponding to their specific IC₅₀, as determined by the clonogenic assay. After 8 h and 48 h of platinum drug exposure with or without radiation treatment, both adherent and floating cells were washed and allowed to grow for an additional 48 h in culture medium. Cells were harvested by trypsinization, pooled with the culture medium that contained floating cells, and collected by centrifugation (3 min, 1300 rpm). The cell pellets were resuspended in 0.5 ml of PBS and were fixed with 1.5 ml of ice-cold 95% ethanol. Before analysis, 100 μl of FBS were added. The cell pellets were re-suspended in 2 ml PBS and were collected by centrifugation (3min, 1300 rpm). The 200 μl of staining solution containing 0.5 mg/ml of RNase A and 5 μg/ml of propidium iodide were added, and the suspension was incubated at 37°C for 30 min. The samples were analyzed using FACScan (Becton Dickinson). The cells in the subdiploid (sub-G₁/G₀) region of the histogram are classified as death cells in apoptosis assay. For each sample 10,000 events were analyzed. These analyses were performed on at least three separate cultures.

Statistical analysis. Results were expressed as the mean±SD of three experiments performed in triplicate. p<0.05 was considered statistically significant (two-tailed paired Student's t-test).