Phenethyl isothiocyanate and paclitaxel synergistically enhanced apoptosis and alpha-tubulin hyperacetylation in breast cancer cells

As described in previous reports [33‐35], PEITC (phenethyl isothiocyanate) was purchased from LKT Labs and dissolved in 70% methanol and 30% deionized water to a stock concentration of 10 mM. Paclitaxel (taxol) powder (Sigma Chemical Co.) was dissolved in DMSO and stored as a stock concentration of 200 nM.

Maintenance and culture of the MCF7 and MDA-MB-231 (MB) cell lines were described in a prior report [37]. Briefly, the cells were seeded at 0.4 × 10⁶ per ml and 0.2 × 10⁶ per ml, respectively, of PRMI-1640 medium supplemented with 10% heat-inactivated fetal bovine serum, 100 IU of penicillin/ml and 100 μg of streptomycin/ml, and maintained at 37°C in a humidified atmosphere containing 5% CO₂. At the specified time points, the cells were harvested. Cell number and viability were determined from at least triplicate cultures by the trypan blue exclusion method.

MCF and MB cells were treated with PEITC and/or paclitaxel at various concentrations for 48 hours. The cell lysates were used for Western blot analysis as described previously [38, 39]. The protein content of the lysates was determined using the BioRad Protein Assay Kit (BioRad, Hercules, CA), with a BSA standard. The antibodies against the following proteins were used for immunoblotting: PARP-1, BCL-2, Bax, Cdk-1, Cyclin B1, α-tubulin, β-tubulin, β-actin, acetyl-α-tubulin, HDAC6, acetyl-H3, and Acetyl-H4 [34, 35, 40]. Secondary antibodies were chosen according to the primary antibodies used (goat anti-rabbit or anti- mouse IgG antibody linked to HRP, Santa Cruz). The proteins were visualized through the ECL system. The protein was quantified using the β-actin protein as the loading control.

Immunostaining of cells for confocal immunofluorescence microscopy was done according to the published methods [35]. Briefly the MCF and MB cells grown on chamber slides were treated for 48 hours without or with PEITC, the cells were then fixed, permeabilized, blocked in BSA and incubated with a mouse anti-acetyl-α-tubulin (Sigma-Aldrich) for 1 h. A fluorescin-conjugated goat anti-mouse IgG was used as secondary antibody. The DNA was counterstained with propidium iodide (PI) to visualize the nuclei of the cells. Images were captured using an MRC 1024 ES confocal laser scanning microscopy system.

Alpha-tubulin has been shown to be acetylated by HDAC6 [41]. When the cells were treated with the combination of PEITC and taxol, the acetylation of alpha-tubulin was significantly increased in both MCF and MB cells in comparison with that in single agent treated cells (Figure 1). When the acetylation level was corrected for the amount of total alpha-tubulin present in the specimen, there was a 16% and 28% respective increase in the specific acetylation level (SAL) of acetylated alpha-tubulin (acetyl-alpha-tubulin per unit of total alpha-tubulin) in MCF cells treated with PEITC or taxol alone (Figure 1). There was a 167% increase in SAL in MCF cells treated with both PEITC and taxol. Therefore, the combination led to a 10.4-fold and 5.96-fold increase in SAL over single agent PEITC and taxol, respectively. This synergistic effect on acetylation of alpha-tubulin was also seen in MB cells (Figure 1). Interestingly, taxol alone also enhanced acetylation of alpha-tubulin in both cell lines. The combination also decreased expression of beta-tubulin more than each agent alone.

To directly visualize the activity of PEITC on breast cancer cells in live cell culture, we next studied the level and distribution of acetylated alpha-tubulin by immunostaining. The cells were visualized with confocal fluorescent microscopy. The cytoplasmic level of acetylated alpha-tubulin clearly increased in both MCF and MB cells after treatment with 5 μM of PEITC for 48 hours, which can be directly visualized under confocal fluorescent microscope (Figure 2).

Cyclin B1 and CDK1 are major cell cycle regulatory proteins for the G2 to M phase progression [42]. To explore the involvement of the major cell cycle regulatory proteins, the level of cyclin B1 and CDK1 expression was studied. Their expressions were characterized with Western blotting. When compared with single agent PEITC and taxol, the combination of both agents reduced the expression of CDK1 more significantly than either agent alone (Figure 3). In the mean time, the cyclin B1 expression was minimally decreased, indicating a less significant effect from the treatment.

Bax and Bcl-2 have opposing effects on apoptosis. Bax promotes apoptosis while Bcl-2 is an anti-apoptosis protein. The levels of the two proteins in the breast cancer cell lines were examined through Western blotting analysis. When compared with single agent PEITC and taxol, the combination of both agents reduced Bcl-2 expression and increased Bax expression more than either agent alone (Figure 4).

PARP proteins are important downstream components of the apoptosis pathways. Cell cycle arrest usually triggers the apoptosis machinery which leads to cellular apoptosis and cell death. The PARP protein cleavage in MCF and MB cells was examined. When compared with single agent PEITC and taxol, the combination of both agents increased the PARP-1 cleavage (thus an increase in the degradation products as described [43]) more than either agent alone in both cell lines (Figure 4).