RPMI, DMEM, and FBS were purchased from Flow Laboratories (Milan, Italy). Tissue culture plasticware was from Microtech (Naples, Italy). Rabbit antiserum raised against caspase 9 and monoclonal antibodies (mAb) raised against caspase 3 and caspase 7 were purchased from Enzo Life Sciences (Florence, Italy). Hydroethidine conjugated secondary antibody was purchased from Sigma-Aldrich (Italy). Mouse antiserum raised against α−tubulin was purchased by Calbiochem (Merck KGaA, Darmstadt, Germany). 5-FU, Doxorubicin and were Levofolene were a gift of Dr. Gaetano Facchini (I.N.T. ‘Pascale’, Naples, Italy).

The rat cardiocytes (H9c2) cell line and the human colon adenocarcinoma (HT-29) cell line obtained from the American Type Tissue Culture Collection, Rockville, MD, grow in DMEM and RPMI1640, respectively, supplemented with heat inactivated 20% FBS, 20 mM HEPES, 100 U/ml penicillin, 100 mg/ml streptomycin, 1% L-glutamine and 1% sodium pyruvate. Both cell lines were grown in a humidified atmosphere of 95% air/5% CO₂ at 37 °C.

Proliferation of H9c2 and HT-29 cell lines was performed in the presence of 5-FU and Doxorubicin (DOXO) in presence or not of Levofolene (LF), by MTT assay as previously described 28.

H9c2 and HT-29 cell lines were grown for 48 h with or without DOXO or 5-FU in presence or not of LF at 37°C. For cell extract preparation, the cells were washed twice with ice-cold PBS, scraped and centrifuged for 30 min at 4°C in 1 ml of lysis buffer (1% Triton, 0.5% sodium deoxycholate, 0.1 NaCl, 1 mM EDTA, pH 7.5, 10 mM Na₂HPO₄, pH 7.4, 10 mM PMSF, 25 mM benzamidin, 1 mM leupeptin, 0.025 units/ml aprotinin). Equal amounts of cell proteins were separated by SDS-PAGE, electrotransferred to nitrocellulose and reacted with the different antibodies. Blot were then developed using enhanced chemiluminescence detection reagents (SuperSignal West Pico, Pierce) and exposed to x-ray film. All films were scanned by using Quantity One software (BioRad laboratories, Hercules, CA).

Annexin V-FITC (fluorescein isothiocyanate) was used in conjunction with a vital dye, Propidium Iodide (PI), to distinguish apoptotic (Annexin V-FITC positive, PI negative) from necrotic (Annexin V-FITC positive, propidium iodide positive) cells. Briefly, cells were incubated with Annexin-V–FITC (MedSystems Diagnostics, Vienna, Austria) and propidium iodide (Sigma, St. Louis, MO, USA) in a binding buffer (10 mM Hepes, pH 7.4, 150 mM NaCl, 5 mM KCl, 1 mM MgCl₂, 2.5 mM CaCl₂) for 10 min at room temperature, washed and resuspended in the same buffer. Analysis of apoptotic cells was performed by flow cytometry (FACScan, Becton Dickinson). For each sample, 2 × 10⁴ events were acquired. Analysis was carried out by triplicate determination on at least three separate experiments.

The cells were seeded in 6-multiwell plates at the density of 3 × 10⁵ cells/well. After 24 h incubation at 37 °C the cells were treated for different time with the IC₅₀s of 5-FU and DOXO. The oxidative stress was analysed by Hydroethidine (HE) staining after 48 h of treatment. Hydroethidine is used as a vital dye in fluorescence assays that operates as a probe for measurement of O₂⁻. The dye enters the cells freely and is dehydrogenated to fluorescent ethidium bromide by O₂⁻. Briefly, the cells were incubated for 1 h at the end of treatment with 20 ng/ml Hydroethidine stock solution (2,5 mg/ml). At the time of processing the cells were scraped, washed twice with PBS and the pellet was resuspended in 1 ml PBS. The dye accumulation was analysed by FACScan flow cytometer (FACScan, Becton Dickinson) by the CellQuest software. For each sample, 2 × 10⁴ events were acquired. Analysis was carried out by triplicate determination on at least three separate experiments.

All data are expressed as mean + SD. Statistical analysis was performed by analysis of variance (ANOVA) with Neumann-Keul's multiple comparison test or Kolmogorov-Smirnov where appropriate.

We studied the effect of increasing concentrations of DOXO and 5-FU in presence or not of LF on growth inhibition of HT-29 and H9c2 cells by MTT assay as described in “Materials and Methods”. We have found a dose and time-dependent growth inhibition in both cell lines. In details, the IC₅₀ (50% inhibitory concentration) value of 5-FU was 4 μM and 400 μM in HT29 and H9c2, respectively (Figure 1 and Table 1). Moreover, LF potentiated growth inhibition induced by 5-FU. In fact, IC₅₀ of HT-29 and H9c2 cells was 2 μM and 43 μM, respectively. These results suggest, as expected, that the colon cancer cell line HT29 was more sensitive to 5-FU than H9c2 normal cells (Table 1). Interestingly, these concentrations of 5-FU can be reached in vivo after the routinely used ways of administration of this agent in the clinical practice 34.

On the other hand, H9c2 cells appeared to be more sensitive to DOXO than HT-29. In fact, the IC₅₀ of DOXO was 0.12 μM and 0.31 μM on HT-29 and H9c2, respectively (Figure 1).

Thereafter, we have evaluated the effects of the different treatments in inducing apoptosis, assessed by FACS analysis after double labelling with Annexin V and PI. We have found that the treatment with DOXO induced apoptosis in only about 8% of H9c2 cell population (Figure 2 and Table 2), while the treatment with 5-FU alone induced apoptosis in about 38% of H9c2 cell population compared to 5% of untreated cells as demonstrated with FACS analysis. Moreover, when the cells were treated with 5-FU and exposed to LF about 45% apoptosis was found. Only about 17% and 8% apoptosis was induced by DOXO and 5-FU, respectively in HT-29 cell line (Figure 2 and Table 2). Therefore, DOXO and 5-FU caused antiproliferative effects in cardiocytes and tumour cells with different mechanisms.

* In bold: significant changes.

To evaluate the intracellular levels of ROS, HT-29 and H9c2 cells were incubated with dihydroethidine followed by FACS analysis of the oxidative product, ethidium, which emits red fluorescence. The mean fluorescence intensity (MFI) corresponds to ROS levels and to intracellular oxidative stress due to superoxide anion (O²⁻) generation induced by their presence.

In H9c2 cells, 5-FU caused an about 1.5-fold increase of MFI reaching an increase of about 2-fold of MFI with the addition of LF indicating a potentiation of oxidative effects (Figure 3 A,B). In the same experimental conditions, we observed an about 3-fold increase of MFI induced by DOXO treatment. In HT29 cells, LF did not potentiate the increase of MFI induced by 5-FU alone that was of about 2-fold while DOXO induced an about 3-fold increase of MFI. Therefore, the oxidative stress induced by DOXO was more potent than that one caused by 5-FU in both cancer cells and cardiocytes. Moreover, LF potentiated the oxidative stress induced by 5-FU only in cardiocytes and not in colon cancer cells.

In order to characterize the apoptosis induced in these cell lines we have evaluated if the different treatments induced the activation of mediators of the execution phase of apoptosis.

We have evaluated the cleavage and the consequent activation of both caspase 9 and 3 with western blotting using specific antibodies that recognize only the intact forms of the two enzymes. We have found that 5-FU increased the cleavage of caspase 3 in H9c2 cells and the latter was potentiated in presence of LF. These effects were paralleled by a decrease of pro-caspase 9 expression (activation index). On the other hand, DOXO increased the cleavage of caspase 3 and 9 after 24 h from the beginning of treatment but the latter returned to basal level after 48 h (Figure 4). Moreover, the different treatments caused no significant changes of the levels of pro-caspase 3 and 9 in HT29 cell line (Figure 5).

These results were consistent with the data derived from FACS analysis; in fact, the treatment with 5-FU and LF induced a stronger apoptotic effect on cardiocytes cell line if compared with that one recorded in colon adenocarcinoma cell line.

In order to study the role of the oxidative stress in the induction of apoptosis by 5-FU in cardiocytes we have exposed H9c2 to the scavenger agent N-acetyl cysteine (NAC) and we have evaluated the effects on apoptosis and intracellular ROS. The addition of NAC alone to H9c2 cells had no effects on apoptosis and intracellular ROS (Figure 6A, B respectively). On the other hand, the addition of NAC to either 5-FU alone or in combination with LF completely abrogated the effects of both on apoptosis and increase in the levels of ROS (Figure 6A, B respectively). We have also used H₂O₂ as positive control and we have found that the addition of 200 μM H₂O₂ to H9c2 cells caused an about 40% apoptosis with an about 2-fold increase of intracellular ROS and that these effects were again abrogated by the concomitant administration of NAC (Figure 6A, B respectively).

These results strongly suggested that apoptosis induced by 5-FU in cardiocytes is likely due to the increase in intracellular ROS.