The mechanism of methylselenocysteine and docetaxel synergistic activity in prostate cancer cells

Abstract

The study was designed to evaluate the combination treatment of methylselenocysteine (MSeC) and docetaxel and to delineate the underlying mechanism associated with observed in vitro synergy between MSeC and docetaxel in prostate cancer cells. Cells were treated with different concentrations and schedules (concurrent or sequential) of MSeC and docetaxel alone or in combination. Cell growth/death was assessed with sulforhodamine B assay, trypan blue assay, and time-lapse video. Loewe synergism/antagonism model was used to determine whether the combination effect was additive, synergistic, or antagonistic. Apoptosis and caspase-3 activity were evaluated with cell death ELISA assay and caspase activity assay, respectively. Synergy between MSeC and docetaxel was further assessed in the presence and absence of z-VAD-fmk, a pan-caspase inhibitor. Effect of MSeC and docetaxel alone or in combination on the cellular expression of the antiapoptotic protein survivin was measured with Western blot analyses. Pretreatment with MSeC was crucial to enhance docetaxel antitumor activity. The enhanced antitumor activity of the sequential combination treatment of MSeC and docetaxel (MSeC/docetaxel) was highly synergistic. Apoptosis increased after MSeC/docetaxel, compared with each drug alone or concurrent treatment. Pretreatment with z-VAD-fmk converted the synergy into antagonism, suggesting that the synergy is caspase-dependent apoptosis. The survivin level was down-regulated following MSeC/docetaxel treatment when compared with each drug alone. In conclusion, pretreatment with MSeC was essential to markedly sensitize cells to docetaxel. The synergy between MSeC and docetaxel in C2G prostate cancer cells is associated with increased level of caspase-dependent apoptosis and decreased level of survivin.

Figure 1

The effects of MSeC and docetaxel, alone or in combination, on cell growth and cell death. Cells were treated with MSeC alone for 24 h (A)or docetaxel alone for 2 h (B) and incubated in drug-free medium for 5 d. Cell growth was assessed with the total protein sulforhodamine B assay. Pretreatment of cells with MSeC for 24 h at concentration that inhibits 25% of cell growth (IC₂₅) followed by multiple concentrations of docetaxel resulted in enhancement of docetaxel inhibition of cell growth (B). C, cells were subjected to a concurrent treatment of MSeC and docetaxel with the same MSeC concentration (IC₂₅). The concurrent treatment did not enhance docetaxel inhibition of cell growth, which suggests that enhancement of docetaxel inhibition of cell growth is MSeC schedule dependent. D, cells were treated with IC₂₅ of MSeC alone for 24 h, IC₉₀ of docetaxel alone for 2 h, sequential combination of MSeC for 24 h followed by docetaxel for 2 h, and concurrent combination of MSeC and docetaxel for 2 h. After staining and normalizing to untreated control, pretreatment with MSeC (sequential combination) significantly increased percentage of cells admitting trypan blue when compared with each drug alone or with the concurrent combination treatment. *, P < 0.05, significantly different when compared with all other treatment groups.

Figure 2

Cell death assessments after the combination treatment of MSeC/docetaxel. Cells were treated with MSeC and docetaxel alone or in combination, stained with trypan blue, and evaluated daily for dye admission/exclusion. A, the sequential combination significantly increased (P < 0.05) percentage of cells admitting trypan blue when compared with each drug alone, indicating more cell death. B, cells were continuously observed with time-lapse video. 1, still digital picture of cells after 24 h of MSeC (IC₂₅) treatment, before adding docetaxel, showing normal profile of cells. 2, picture of the same field, 5 d after treatment with 2 h docetaxel (IC₉₀), showing that >95% of cells exhibited marked shrinkage, loss of cells, and breakage into smaller parts (apoptotic bodies), which is consistent with cell death by an apoptotic mechanism. *, P < 0.05, significantly different when compared with all other treatment groups.

Figure 3

Synergistic effect between MSeC and docetaxel. A SYNFIT custom graph shows the degree of bowing of the additive line corresponding with the absolute value of antagonism/synergism variable α. *, D₁ / D_X1 is the dose of MSeC in combination over the dose of MSeC when applied as a drug alone that achieves an X effect. **, D₂ / D_X2 is the dose of docetaxel in combination over the dose of docetaxel when applied as a drug alone that achieves an X effect. The inward bowing of the straight additive line is an indication of the combination synergistic effect that achieves 90% or 50% inhibition of cell growth by the combination treatment. The sequential combination treatment was highly synergistic, indicated by the degree of inward bowing.

Figure 4

The sequential combination treatment of MSeC and docetaxel induces caspase-3 activity and apoptotic cell death. Cells were treated with MSeC alone, docetaxel alone, and sequential combination of MSeC/docetaxel. The sequential combination treatment induces caspase-3 activity immediately (A) and at different time points (B) after drug treatments when compared with each drug alone treatment. C, the sequential combination treatment significantly induced apoptotic cell death when compared with each drug alone or pretreatment with z-VAD-fmk. D, treated cells were stained with trypan blue to assess cell death. Pretreatment with z-VAD-fmk significantly reduced percentage of cells admitting the dye, linking apoptosis to cell death. *, P < 0.05, significantly different when compared with all other treatment groups.

Figure 5

MSeC and docetaxel synergy is caspase-dependent apoptosis synergy. Cells were assessed for the combination synergy with pretreatment of z-VAD-fmk using SYNFIT generated graph of Hill and Loewe additivity models. *, D₁ / D_X1 is the dose of MSeC in combination over the dose of MSeC when applied as a drug alone that achieves an X effect. **, D₂ / D_X2 is the dose of docetaxel in combination over the dose of docetaxel when applied as a drug alone that achieves an X effect. After pretreatment with z-VAD-fmk, the synergistic inward bowing of the combination treatment lines (seen without z-VAD-fmk pretreatment in Fig. 3) changed to antagonistic outward bowing lines in all concentrations. This conversion from synergy to antagonism with blocking of all caspases through pretreatment with z-VAD-fmk suggests that the combination synergy is caspase-dependent apoptosis.

Figure 6

The sequential combination treatment down-regulates survivin expression. Cells treated with two different concentrations of docetaxel alone or in combination [IC₉₀ (A) or IC₁₀ (B) for 2 h] with a fixed concentration of MSeC (IC₂₅ for 24 h). Survivin expression was assessed by Western blot analysis and loaded samples were numbered in both treatments from 1 to 4. A1 and B1, samples of untreated control; A2 and B2, samples of MSeC alone at IC₂₅; A3 and B3, samples of docetaxel alone at IC₉₀ and IC₁₀, respectively; A4 and B4, samples of MSeC at IC₂₅ in combination with docetaxel at IC₉₀ and IC₁₀, respectively. Down-regulation of survivin was associated with the combination treatment of MSeC and docetaxel at a concentration of docetaxel that achieved synergy (IC₉₀) but not with a docetaxel concentration that achieved additivity (IC₁₀).