Rapamycin potentiates the effects of paclitaxel in endometrial cancer cells through inhibition of cell proliferation and induction of apoptosis

Abstract

Mammalian target of rapamycin (mTOR) inhibitors modulate signaling pathways involved in cell cycle progression, and recent phase II trials demonstrate activity in patients with endometrial cancer. Our objective was to examine the effects of combination therapy with rapamycin and paclitaxel in endometrial cancer cell lines. Paclitaxel inhibited proliferation in a dose-dependent manner in both cell lines with IC(50) values of 0.1-0.5 nM and 1-5 nM for Ishikawa and ECC-1 cells, respectively. To assess synergy of paclitaxel and rapamycin, the combination index (CI) was calculated by the method of Chou and Talalay. Simultaneous exposure of cells to various doses of paclitaxel in combination with rapamycin (1 nM) resulted in a significant synergistic anti-proliferative effect (CI <1, range 0.131-0.920). Rapamycin alone did not induce apoptosis, but combined treatment with paclitaxel increased apoptosis over that of paclitaxel alone. Treatment with rapamycin and paclitaxel resulted in decreased phosphorylation of S6 and 4E-BP1, two critical downstream targets of the mTOR pathway. Rapamycin decreased hTERT mRNA expression by real-time RT-PCR while paclitaxel alone had no effect on telomerase activity. Paclitaxel increased polymerization and acetylation of tubulin, and rapamycin appeared to enhance this effect. Thus, in conclusion, we demonstrate that rapamycin potentiates the effects of paclitaxel in endometrial cancer cells through inhibition of cell proliferation, induction of apoptosis and potentially increased polymerization and acetylation of tubulin. This suggests that the combination of rapamycin and paclitaxel may be a promising effective targeted therapy for endometrial cancer.

Conflict of interest statement

CONFLICT OF INTEREST STATEMENT

We, the authors of this manuscript, have no financial or personal relationships to disclose that could inappropriately influence or bias this work.

Figures

Figure 1

Effect of paclitaxel with and without rapamycin on proliferation of endometrial carcinoma cells. Ishikawa and ECC-1 cells were cultured in the presence of varying concentrations of paclitaxel and rapamycin. Rapamycin enhances sensitivity to paclitaxel after 48 hours of exposure in both cell lines. The relative growth of cells was determined by MTT. The IC50 values of paclitaxel with rapamycin (1nM) were 0.1–0.5 nM (p = 0.0004 – 0.0097) and 0.01–0.001 nM (p = 0.0001–0.0357) for Ishikawa and ECC-1 cells, respectively. The results are shown as the mean ± SE of triplicate samples and are representative of three independent experiments.

Figure 2

Paclitaxel and rapamycin in combination increased apoptosis compared to paclitaxel alone. Rapamycin alone did not induce apoptosis. The (A) Ishikawa and (B) ECC-1 cell lines were grown for 24 hours and then treated with the indicated concentrations of rapamycin, paclitaxel or paclitaxel in combination with 1 nM rapamycin. Apoptosis was assessed using an antibody to dsDNA. Data shown are representative of at least two independent experiments (* indicates statistically significant difference).

Figure 3

The effect of paclitaxel and rapamycin on phosphorylation of S6 in (A) Ishikawa and (B) ECC-1 cells. The cells were treated with rapamycin (1 nM), paclitaxel (1 nM) or both in combination for 24–48 hours. Rapamycin alone and in combination with paclitaxel completely abolished phosphorylation of S6. Treatment with paclitaxel alone also reduced phosphorylation of the S6 protein in Ishikawa (C) and ECC-1 cells (D). This effect was best seen after 72 hours of exposure to paclitaxel. Phosphorylated S6, pan-S6 and β-actin were determined by Western immunoblotting.

Figure 4

The effect of paclitaxel and rapamycin on phosphorylation of 4E-BP1 in (A) Ishikawa and (B) ECC-1 cells. The cells were treated with rapamycin (1 nM), paclitaxel (1 nM) or both in combination for 24–48 hours. Rapamycin alone and in combination with paclitaxel decreased phosphorylation of 4E-BP1 and pan-4E-BP1. Treatment with paclitaxel alone had no effect on the phosphorylation of 4E-BP1 in either cell line (C & D). Phosphorylated 4E-BP1, pan-4E-BP1 and α-tubulin were determined by Western immunoblotting.

Figure 5

Paclitaxel did not affect hTERT mRNA expression in the (A) Ishikawa and (B) ECC-1 cell lines. Treatment with rapamycin alone (p = 0.04 for Ishikawa cells; p = 0.0067 for ECC-1 cells) and in combination with paclitaxel (p = 0.05–0.06 for Ishikawa cells; p = 0.01–0.04 for ECC-1 cells) resulted in decreased hTERT mRNA expression. Both cell lines were cultured for 24 hours and then treated with the indicated concentrations of paclitaxel alone or in combination with rapamycin (1 nM) for 48 hours. hTERT expression was determined by real-time PCR. The results are shown as the mean ± SE of two independent experiments (* indicates statistically significant difference).

Figure 6

Alterations in cellular microtubule structures resulting from exposure to paclitaxel and rapamycin in (A) Ishikawa and (B) ECC-1 cell lines. Microtubules were visualized by immunoflourescence using a primary antibody for α–tubulin. The combination of paclitaxel with rapamycin appeared to potentiate the effect of paclitaxel alone on α-tubulin polymerization and organization in both cell lines.

Figure 6

Alterations in cellular microtubule structures resulting from exposure to paclitaxel and rapamycin in (A) Ishikawa and (B) ECC-1 cell lines. Microtubules were visualized by immunoflourescence using a primary antibody for α–tubulin. The combination of paclitaxel with rapamycin appeared to potentiate the effect of paclitaxel alone on α-tubulin polymerization and organization in both cell lines.

Figure 7

The effect of paclitaxel and rapamycin on tubulin acetylation by Western immunoblotting. Ishikawa and ECC-1 cells were treated with paclitaxel (0.1uM), rapamycin (1 uM) or both in combination for 24 hours. (A) Increased tubulin acetylation was found in cells treated with both paclitaxel and rapamycin. (B) As quantified by densitometer analysis, treatment with paclitaxel and rapamycin increased acetylated tubulin expression by 3.1 and 4.0 fold for the Ishikawa and ECC-1 cell lines, respectively. Paclitaxel alone increased tubulin acetylation by 1.5 and 1.3 fold for the Ishikawa and ECC-1 cell lines, respectively. Rapamycin alone had no effect on acetylated tubulin. No effect was seen on α-tubulin expression.