3.1. Tacca A orγ-radiation cause G2/M accumulation
Cell cycle progression is known to be inhibited by diverse microtubule targeting agents or γ-radiation. The effects of tacca A or γ-radiation on the cell cycle progression of SCC4 cells was evaluated to determine whether these treatments caused G2/M accumulation. In control, untreated SCC4 cells, 47.2% of the population were in G1, 27.2% in S phase, and 25.6% were in G2/M (). Treatment of cells with either γ-radiation or tacca A caused a dose dependent accumulation of cells with 4 N DNA content, the G2/M population, within 24 h (). Although concentrations up to 1 μM tacca A did not significantly affect cell cycle distribution, 2 μM tacca A doubled the number of cells in G2/M from 25.6% to 58.0%, with corresponding decreases in the G1 and S phase populations of cells (). Increasing the concentration of tacca A to 4 μM further increased the percentage of cells in G2/M to 82.2%. A dose of 2 Gy of γ-radiation had no effect on cell cycle distribution and a slight increase in cells in the G2/M population was initiated with a 4 Gy dose (). A near doubling of the G2/M population from 25.6% to 49.1% was achieved with 6 Gy of γ-radiation, mostly due to a decrease in the S phase population. 10 Gy of γ-radiation further increased the G2/M population of cells to 69.9%. These data confirm that SCC4 cells accumulate in G2/M in response to either tacca A or ionizing radiation treatment.
Dose-dependent effects of tacca A and γ-radiation on cell cycle distribution. The cell cycle distribution of propidium iodide stained SCC4 cells was analyzed 24 h after treatment with tacca A or radiation.
Propidium iodide-based cell cycle analysis cannot distinguish the G2
and M phases of the cell cycle, but most adherent cells, including SCC4 cells, appear flat in interphase but undergo a RhoA-dependent change in the actin cytoskeleton during mitosis to become noticeably more spherical [20
]. Therefore, the G2
and M phases of the cell cycle can be easily distinguished by light microscopy. The approximate percentage of cells that were in mitosis after treatment with γ-radiation or tacca A was determined by visually examining the morphology of cells prior to their preparation for flow cytometry. The percentage of spherical mitotic cells observed upon tacca A treatment roughly correlated with the percentage of cells with 4N DNA content as determined by flow cytometry. This finding supports previous reports that the taccalonolides arrest cells in mitosis [16
]. In contrast, γ-radiation did not noticeably increase the percentage of spherical cells even at 10 Gy, a concentration that tripled the accumulation of G2
/M cells. This is consistent with reports that γ-radiation leads to G2
arrest of cells, like SCC4, that express a mutant form of p53 [21
]. Therefore, although both tacca A and γ-radiation cause a dose-dependent G2
/M accumulation of SCC4 cells, tacca A causes mitotic arrest while cells appear to accumulate in G2
in response to γ-radiation.
3.2. Tacca A and γ-radiation inhibit short-term cell viability in an additive manner
The cytotoxic effects of tacca A and radiation were evaluated alone and in combination using a high-throughput fluorescence microscopy assay. The number of live (green) and dead (red) cells were counted following treatment with tacca A, paclitaxel, γ-radiation or a combination of drug and γ-irradiation. The assay clearly differentiates live and dead cells () and the high throughput functionality allowed the evaluation of 10,000 – 22,000 cells for each treatment group. Consistent with the colorimetric readout of the assay shown in , a histogram showing the raw data of the effects of tacca A treatment alone is presented in . The histogram retains the green/red coloring indicating live/dead cells remaining on the plate. The percentage of viable cells is a conservative readout, since any cells lost from the plate due to cell death during the experiment are not detected. We calculated a doubling time of 26 hrs for untreated cells for the duration of the assay, which means that cells were exposed to drug for one population doubling before being subjected to radiation and that viability was analyzed after an additional population doubling. Tacca A alone caused a dose dependent decrease in viable cells from 98% in untreated controls to 91% viability at 0.75 μM, 80% at 1.25 μM and 66% at 1.75 μM tacca A (; ). A dose-dependent increase in the number of dead cells retained on the plate was also observed (). The effects of paclitaxel on cell viability were also determined to allow comparisons between the two microtubule stabilizers. In untreated wells, live viable cells represented 98% of the population while treatment with 3 nM paclitaxel resulted in 81% viable cells (). As with tacca A, the number of total viable cells remaining after paclitaxel treatment diminished in a dose-dependent manner (). Ionizing radiation alone also caused cell death, with cell viability decreasing to 92% and 80% at 6 Gy and 10 Gy respectively (). The magnitude of the individual effects of tacca A, paclitaxel or radiation treatment on cell viability was used to select concentrations of each treatment to evaluate in combination. Concentrations of tacca A or paclitaxel and doses of radiation that caused sub-maximal cell death, retaining 65 - 90% cell viability after treatment with a single modality, were selected. This defined an optimal range to detect additive or synergistic actions of combination treatments.
Fig. 2 Effects of γ-radiation and tacca A in the short term viability assay. Representative image of live (green) and dead (red) cells (A). Number of live (green) and dead (red) cells following treatment with tacca A alone (B). Percentage of live cells (more ...)
Percent cell viability of SCC4 cells when treated with single agents in the short term viability assay.
Concentration ranges of 0 – 1.75 μM tacca A, 0–6 nM paclitaxel and radiation doses of 6 and 10 Gy were selected to optimally detect additive or potential synergistic actions of combination treatments. shows the effects of tacca A treatment alone and in combination with 6 or 10 Gy of radiation. Within each concentration of tacca A the cytotoxic effects of radiation are observed and the ability of the combination to decrease cell viability across the range of concentrations of tacca A is seen. The combination of 1.75 μM tacca A and radiation caused a decrease in cell viability from 66% with tacca A alone to 52% at 6 Gy and 44% in combination with 10 Gy of radiation (). Similar effects were observed with paclitaxel ().
The data were evaluated further to determine whether the effects of combining a microtubule stabilizer with radiation cause additive loss of cell viability or indicated a potential synergistic effect. The predicted additive effects of tacca A and radiation on cell viability were calculated for each combination from their individual effects and compared to the actual effects measured when the 2 treatment modalities were combined. This analysis indicates that various combinations of γ-radiation and tacca A have effects that are statistically indistinct from their predicted additive values (). The predicted additive effects between paclitaxel and γ-radiation were also determined and compared to measured effects. Additive effects between paclitaxel andγ-radiation were observed with almost every combination treatment (). These analyses suggest that the combination of tacca A or paclitaxel with radiation cause additive effects on SCC4 cell viability as measured with the high-throughput, short term viability assay and provides no indication of a potential synergistic effect of combining either tacca A or paclitaxel with radiation.
The advantage of the short-term high throughput assay is its robustness due to the large number of cells that can be counted in each experimental group. A limitation of this assay is that there is no way to evaluate the fate of individual cells that are lost from the plate during the course of the 48 h experiment. An example can be seen in , where 22,269 cells were evaluated in untreated controls yet the number of cells decreased to 15,711 with 1.75 μM tacca A treatment. The difference in total cell number upon treatment could be due to cell death and/or an increase in mitotic cells, which are more susceptible to being dislodged from the plate. Another potential shortcoming of this analysis is that radiation induced cell death often requires numerous mitotic events to allow chromosomal damage to accumulate to a point that is lethal, which will not be reflected in this short-term assay where cells have only gone through one mitotic event on average between irradiation and viability measurements. To address these issues, the effects of combining tacca A and radiation in a longer term clonogenic assay were also studied.
3.3. The taccas and γ-radiation have additive effects on colony formation
The colony formation assay was used to evaluate the long-term effects of tacca A and γ-radiation on SCC4 cell growth and viability when used alone or in combination. In untreated controls an average of 165 colonies formed during the assay (, top left panel). The concentration dependent effects of tacca A treatment alone on colony formation were first determined (, solid line). Concentrations of tacca A up to 125 nM did not inhibit the colony forming ability of the SCC4 cells (). However, colony formation was inhibited by 28% with 150 nM tacca A and this inhibition is clearly visible in the treated plates (, top right panel). A 49% inhibition of survival was observed with 175 nM tacca A and 76% inhibition with 200 nM tacca A (, solid line; ).
Fig. 3 Effects of γ-radiation and tacca A on colony formation. (A) Images of crystal violet-stained colonies formed after treatment of SCC4 cells with 150 nM tacca A, 2 Gy γ-radiation or a combination of the two treatments. The percentage of (more ...)
Percent cell viability of SCC4 cells when treated with single agents in the clonogenic assay.
The effects of radiation treatment alone on survivability were also evaluated and the data presented as the solid line of . A 1 Gy dose inhibited colony formation 15%, 2 Gy caused 22% inhibition, 4 Gy caused 61% inhibition and a 6 Gy dose inhibited colony formation 89% (). Consistent with the effects of the short-term assay, tacca A or radiation alone inhibit the long-term viability of SCC4 cells as measured by the colony forming assay, albeit both drug and radiation effects are observed at lower doses.
The effects of combinations of tacca A and radiation treatment on colony formation were analyzed. Cells were treated with low concentrations of tacca A 24 h prior to irradiation and colonies counted 12 days afterward. The results show that 2 Gy γ-radiation significantly enhanced the effectiveness of tacca A treatment up to a concentration of 175 nM (, compare solid to dotted lines). Similarly 150 nM tacca A significantly decreased colony formation when combined with doses of 1 or 2 Gy γ-radiation (, compare solid to dotted lines). Not surprisingly, the added benefit of low concentrations of tacca A pretreatment were not statistically significant at higher doses ofγ-radiation since these 4 and 6 Gy doses cause a substantial inhibition of colony formation on their own.
The additive effects of tacca A and radiation treatment in the clonogenic assay were evaluated. The effects of one combination are presented in , where 150 nM tacca A as a single agent decreased viability to 72% of untreated control (top right panel) and 2 Gy γ-radiation decreased viability to 78% when used alone (bottom left panel). The combination, shown in the bottom right panel, caused 50 % viability, demonstrating an additive effect of tacca A and radiation in this assay. The expected additive percent inhibition of colony formation for each combination of tacca A and radiation was calculated by adding together their individual effects with propagated error. The results in show that the actual inhibition of colony formation generated by cells treated with 100 or 150 nM tacca A 24 h prior to exposure with 1 or 2 Gy γ-radiation are not statistically different than the predicted additive values, suggesting that these two treatment modalities have additive effects on cellular viability in the clonogenic assay.
Fig. 4 Effects of γ-radiation and tacca treatment are additive in the clonogenic assay. (A) Tacca A was added to SCC4 cells 24 h prior to γ-radiation exposure. (B) SCC4 cells were exposed toγ-radiation 24 h prior to tacca A addition. (more ...)
The fact that tacca A and radiation treatments have strictly additive and not synergistic effects on cellular viability suggests that tacca A is not a true radiosensitizer. To further analyze the relationship between tacca A and radiation, the order of treatments was reversed such that SCC4 cells were subjected to γ-radiation 24 h prior to tacca A treatment. We found that the combination of these two treatments retained their additivity in the clonogenic assay when the order of administration was reversed. This is demonstrated by the fact that the actual effect on viability of cells irradiated prior to treatment with tacca A is not statistically significant from their predicted additive effects (). This finding supports the hypothesis that tacca A is not strictly radiosensitizing cells, but is instead contributing toward a decrease in cellular viability through a distinct mechanism that is complementary to radiation treatment regardless of the order in which the two treatments are administered. Additional clonogenic experiments using MCF7 cells treated with tacca A prior to γ-radiation () and SCC4 cells treated with tacca E prior to γ-radiation () demonstrated that the additivity between these two treatment modalities is a generalizable property of the taccas in disparate cell lines. Therefore, the data generated with the longer term clonogenic assay are consistent with the results obtained in the short term viability assay in the sense that they both demonstrate that the taccas and γ-radiation have additive effects on cellular viability.