Synergistic inhibition of colon cancer cell growth with nanoemulsion-loaded paclitaxel and PI3K/mTOR dual inhibitor BEZ235 through apoptosis

Abstract

Colon cancer is the third most common cancer in the world, with drug resistance and metastasis being the major challenges to effective treatments. To overcome this, combination therapy with different chemotherapeutics is a common practice. In this study, we demonstrated that paclitaxel (PTX) together with BEZ235 exhibited a synergetic inhibition effect on colon cancer cell growth. Furthermore, nanoemulsion (NE)-loaded PTX and BEZ235 were more effective than the free drug, and a combination treatment of both NE drugs increased the efficiency of the treatments. BEZ235 pretreatment before adding PTX sensitized the cancer cells further, suggesting a synergistic inhibition effect through the phosphatidylinositol-3-kinases/protein kinase B/mammalian target of rapamycin pathway. The 50% inhibitory concentrations for BEZ235 were 127.1 nM and 145.0 nM and for PTX 9.7 nM and 9.5 nM for HCT-116 and HT-29 cells, respectively. When loaded with NE the 50% inhibitory concentrations for BEZ235 decreased to 52.6 nM and 55.6 nM and for PTX to 1.9 nM and 2.3 nM for HCT-116 and HT-29 cells, respectively. Combination treatment with 10 nM NE-BEZ235 and 0.6 nM and 1.78 nM NE-PTX could kill 50% of HCT-116 and HT-29, respectively. The cell death caused by the treatment was through apoptotic cell death, which coincided with decreased expression of anti-apoptotic protein B-cell lymphoma 2. Our data indicate that the combination therapy of PTX with the phosphatidylinositol-3-kinases/protein kinase B/mammalian target of rapamycin dual inhibitor BEZ235 using NE delivery may hold promise for a more effective approach for colon cancer treatment.

Keywords: chemotherapy; combination therapy; nanomedicine; signal pathway inhibitor.

Figure 1

Particle size of NE: the particle size distributions of NE, NE-PTX, and NE-BEZ235 from DLS analysis. Abbreviations: DLS, dynamic light scattering; NE, nanoemulsion; PTX, paclitaxel.

Figure 2

Dose-dependent inhibitory effect of PTX and BEZ235 on colon cancer cells. Notes: (A) MTT assay results on colon cancer cells HCT-116 and HT-29 treated with PTX at various concentrations (in nM). The two cell lines have a similar sensitivity to the drug treatment. (B) MTT assay results of BEZ235 in the two cell lines at different concentrations (in nM). The two cell lines show no differences in sensitivity to the treatment. (C) Combination treatment with a fixed BEZ235 concentration of 25 nM and various PTX concentrations. At 25 nM of BEZ235, both cell lines exhibit an increased sensitivity to PTX treatment. (D) Further increased cell sensitivity to PTX by pretreatment of the cells with BEZ235 for 2 hours followed by adding the same amount of PTX. Abbreviations: MTT, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide; PTX, paclitaxel.

Figure 3

Dose-dependent inhibitory effect of NE-loaded PTX and BEZ235 on colon cancer cells. Notes: The MTT assay results show the effect of NE-PTX and NE-BEZ235 on HCT-116 and HT-29 cells. (A) Effect of NE-PTX. (B) Effect of NE-BEZ235. (C) Effect of the combination treatment with the two drugs. (D) Effect of unloaded NE nanoparticles. Abbreviations: MTT, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide; NE, nanoemulsion; PTX, paclitaxel.

Figure 4

Cell death and morphology changes after BEZ235 and PTX treatment. Notes: (A) White-field images of the cell death caused by PTX and BEZ235 treatment alone or combination treatment after 24 hours. The dead cells and decreased cell numbers were clear in treated groups compared to the controls. (B) Cell morphology changes after 2 hours treatment with PTX or BEZ235 or both. All images were taken using an Olympus IX51 fluorescent microscope at 100× magnification. Abbreviations: NE, nanoemulsion; PTX, paclitaxel.

Figure 5

Cell cycle analysis of HCT-116 and HT-29 cells treated with BEZ235 and PTX. Notes: The flow cytometry result shows the cell cycle analysis of DNA contents labeled by PI, which is shown by the FL2 channel. Single cells were gated by FSC-H versus FSC-A. All other analyses were based on single populations. (A) Analysis on HCT-116 cells. (B) Analysis on HT-29 cells. Abbreviations: FL2-A, PI channel; FSC-A, forward scatter-area; FSC-H, forward scatter-height; PI, propidium iodide; PTX, paclitaxel.

Figure 6

Annexin V apoptosis analysis for the combination treatment of BEZ235 and PTX. Notes: The flow cytometry analysis of apoptotic cell death shows that the cotreatment of BEZ235 and PTX has induced more cell death in (A) HCT-116 cells and (B) HT-29 cells than any single drug treatment at different doses of 2 nM and 4 nM for PTX and 50 nM for BEZ235. Abbreviations: FL1-A, Annexin V staining; FL3-A, propidium iodide staining; LL, lower left; LR, lower right; PTX, paclitaxel; Q4, quarter 4; UL, upper left; UR, upper right.

Figure 7

Bcl-2 Western-blot analysis for the combination treatment of BEZ235 and PTX. Note: Combination treatment of BEZ235 and PTX decreased Bcl-2 protein levels detected by Western blot in both HCT-116 and HT-29 colon cancer cells. Abbreviations: Bcl-2, B-cell lymphoma 2; Tub, human α-tubulin; PTX, paclitaxel.