PC-12 cells were maintained in Dulbecco's Modified Eagle Media (DMEM; Gibco) with 10% calf serum (Turbo calf serum, Invitrogen), 5% horse serum (JRH Biosciences, Victoria, Australia) and 1% penicillin/streptomycin (Invitrogen) at 37°C, in a 5% CO₂ atmosphere. Differentiation into neurons was achieved by seeding cells at a density of 15,000 cells/well in 24-well plates (Falcon Becton Dickinson), on 13 mm glass cover slips (Menzel Glaser, Germany) coated with laminin (Invitrogen) and poly-DL-ornithine (Sigma) in DMEM plus 1% horse serum and 50 ng/mL of nerve growth factor (NGF) (Sigma). The cells were incubated for 72 hours under differentiation conditions, as previously described 20 before use in the neurite outgrowth assays below.

Cisplatin (Sigma) and PXD were provided by Novogen. They were dissolved in DMSO and stocks maintained at -20°C. Working concentrations were diluted in PC12 cell differentiation medium as above. Control conditions contained DMSO diluted as for the drug conditions.

Combination treatments consisted of a 24 hour incubation in differentiation media with various combinations of strong or moderate doses of Cisplatin (20 μg/ml/66.65 μM and 1 μg/ml/3.33 μM) and three doses of PXD (100 nM, 1 μM, 10 μM) as described in the results section. Recovery treatments consisted of treating differentiated cells with either dose of Cisplatin for 24 hours. After this period, the cells were allowed to recover for 24 hours in fresh differentiation media. Finally, three concentrations of PXD were added (10 nM, 100 nM, 1 μM) for a further 24 hours to assess whether PXD could rescue the neurites from the Cisplatin induced damage.

Following the treatment period, cells were washed with PBS, fixed with 4% paraformaldehyde and permeabilised with ice-cold methanol. Neurons were immunostained for the neuronal marker βIII-tubulin (Promega; Madison, Wisconson, USA) and a Cy3-conjugated anti-mouse antibody (Invitrogen) was used to visualise the staining.

Outgrowing projections were considered neurites if they were greater than a cell body width (10 μm), essentially as previously described 21. Images of all neurons in thirty randomly selected fields were digitally captured using an Olympus BX60 fluorescence microscope with a UPlanFl 40x/0.75 objective lens and a Zeiss Axiocam HRc digital camera and Zeiss Axiovision 3.1 software. Neurons in all fields were counted per condition and the number of cells with neurites expressed as a percentage of total cells present and error expressed as the standard error of the mean (sem). Each experiment was conducted in triplicate and the results pooled. Statistical significance of data was analysed using ANOVA followed by the Bonferroni post-hoc test.

Further analysis was conducted on the combination treatments, where the longest neurite per neuron present in each frame was measured using Image J open source software (NIH, USA).

Optimal treatment concentrations Cisplatin that caused neurite damage were determined by treating differentiated PC12 cells for 24 hours with serial dilutions of the drug. The concentration which caused the greatest reduction in the percentage of cells with neurites, without killing the cells, was chosen as a strong dose. A moderate dose was also selected, where the drug reduced neurite outgrowth by approximately 50% of the strong dose. The strong dose of Cisplatin was selected as 20 μg/mL (66.65 μM), which reduced the percentage of cells with neurites by 65.8%, and the moderate dose of 1 μg/mL (3.33 μM) caused a 31.9% reduction in percentage of cells with neurites (Fig. 1a). The chosen concentrations did not produce cytotoxicity (data not shown).

A serial dilution was also conducted for PXD to determine the concentration which would not affect normal growth of the differentiated cells. In addition to the maximal concentration tested that did not affect survival of PC12 cells (1 μM; 3.2 μg/ml), two other concentrations were selected for treatments, one log above (10 μM; 32 μg/ml) and one log below (100 nM; 320 ng/ml). Doses up to 1 μM had no effect on cell death or neurite outgrowth (Fig. 1b), although a 10 μM concentration showed considerable cytotoxicity (data not shown).

To determine whether PXD could block Cisplatin induced neurite toxicity, three different concentrations of PXD were added to cells in combination with Cisplatin at the concentrations determined above to produce strong or moderate neurite toxicity. PXD had no effect on percent neurites at 100 nM or 1 μM but showed significant neurite toxicity at 10 μM (# P < 0.001 compared to no treatment control) (Fig. 2a). This neurite toxicity was exacerbated in combination with Cisplatin, with increased toxicity compared to PXD at 10 μM alone (z P < 0.01 in combination with Cisplatin 1 μg/ml; ## P < 0.001 in combination with Cisplatin 20 μg/ml) (Fig. 2b).

Although in the initial dose response experiments Cisplatin at 1 μg/ml produced a modest neurite toxicity, this was not robustly observed when subsequent experiments were performed (the effect was not significant when all data was analysed using ANOVA with a Bonferroni post-hoc test, although a direct comparison of control versus Cisplatin 1 μg/ml using the t-test was significant; P < 0.02) (Fig. 2b). Consequently, although there appeared to be a slight protection by PXD, this was not significant by ANOVA, although comparison of Cisplatin 1 μg/ml with Cisplatin 1 μg/ml+PXD 1 μM was significant by t-test (P < 0.02). Robust neurite toxicity was observed with Cisplatin at 20 μg/ml, with a 42% decrease in percent neurites compared to control (*** P < 0.001). PXD blocked this neurite toxicity by approximately 50% at 100 nM (* P < 0.01) and 1 μM (** P < 0.001) (Fig. 2b; Fig. 4).

To determine whether there were more subtle effects on neurite toxicity than could be measured by counting the percent of cells with neurite as above, the effect of Cisplatin and PXD on neurite length was examined. The 10 μM concentration of PXD resulted in significant reductions in average neurite length compared to no treatment control, both alone (Fig. 3a) and in combination with Cisplatin (Fig. 3b) (# p < 0.001). While Cisplatin decreased the percentage of cells that had neurites (Figs. 1, 2), it had no significant effect on the average neurite length of the remaining neurites (Fig. 3b). Interestingly, while PDX (100 nM and 1 μM) or Cisplatin alone had no effect on neurite length, the combination of these drugs increased neurite length compared to Cisplatin alone (* p < 0.001), reflecting a blocking of Cisplatin neurite toxicity. Overall, the effect of PXD on blocking Cisplatin effects on neurite outgrowth reflected the observations for the effects on percentage of cells with neurites.

Co-administration of PXD with Cisplatin blocked the Cisplatin-induced neurite toxicity. To determine whether it could have an effect on recovery from Cisplatin induced neurite toxicity by exacerbation, reversal, retardation or facilitation of repair, recovery experiments were performed. When conducting the recovery experiments, the concentrations of PXD tested were decreased by 1 log to 10 nM, 100 nM and 1 μM so that the highest concentration was below the toxic level. Differentiated PC12 cells were incubated with Cisplatin for 24 hrs as above, which was then washed off and the cells allowed to recover for 24 hrs before PXD was added for a further 24 hrs. Addition of PXD at 10 nM, 100 nM or 1 μM had no effect on neurite outgrowth by themselves (Fig. 5a).

After 48 hrs of recovery, Cisplatin showed no neurite toxicity at 1 μg/ml but greater than 50% toxicity at 20 μg/ml (*P < 0.001) compared to the no treatment control, indicating that the cells could not recover from this higher dose by 48 hrs after drug addition. PXD had no significant effect on recovery of neurite outgrowth following 20 μg/ml Cisplatin treatment, although there was a slight enhancement of neurite outgrowth by PXD (1 μM), following Cisplatin at 1 μg/ml (**P < 0.001) (Fig. 5b). Overall, PXD was protective for Cisplatin induced neurite toxicity at concentrations of 100 nM to 1 μM when co-administered but not when administered after toxicity had already occurred.