Co-treatment by docetaxel and vinblastine breaks down P-glycoprotein mediated chemo-resistance

Abstract

Objectives: Chemoresistance remains the main causes of treatment failure and mortality in cancer patients. There is an urgent need to investigate novel approaches to improve current therapeutic modalities and increase cancer patients' survival. Induction of drug efflux due to overexpression of P-glycoproteins is considered as an important leading cause of multidrug resistance. In this study, we investigated the role of combination treatments of docetaxel and vinblastine in overcoming P-glycoprotein mediated inhibition of apoptosis and induction of cell proliferation in human non-small cell lung carcinoma cells.

Materials and methods: Cell proliferation and apoptosis were assessed using MTT assay and DAPI staining, respectively. P-glycoprotein expression was evaluated in gene and protein levels by Real-time RT-PCR and Western blot analysis, respectively.

Results: Combination treatment of the cells with docetaxel and vinblastine decreased the IC50 values for docetaxel from (30±3.1) to (15±2.6) nM and for vinblastine from (30±5.9) to (5±5.6) nM (P≤0.05). P-glycoprotein mRNA expression level showed a significant up-regulation in the cells incubated with each drug alone (P≤0.001). Incubation of the cells with combined concentrations of both agents neutralized P-glycoprotein overexpression (P≤0.05). Adding verapamil, a P-glycoprotein inhibitor caused a further increase in the percentage of apoptotic cells when the cells were treated with both agents.

Conclusion: Our results suggest that combination therapy along with P-glycoprotein inhibition can be considered as a novel approach to improve the efficacy of chemotherapeutics in cancer patients with high P-glycoprotein expression.

Keywords: Chemoresistance; Chemotherapy; H1299 cells; Lung cancer; Verapamil.

Figure 1

Effects of docetaxel and vinblastine alone and in combination on proliferation and apoptotic response in H1299 cells. Panel (A) shows cell viability (%) of the cells after 24 and 48 hrs incubation with vinblastine (0-100 nM) or docetaxel (0-100 nM). Panel (B) shows MTT results of incubation of the cells with combination of docetaxel and vinblastine. The cells were incubated with different concentrations of vinblastine (0-100 nM) and docetaxel (30nM) or different concentrations of docetaxel (0-100 nM) and vinblastine (30 nM) for 48 hrs. Results are expressed relative to the cell density of non-treated cells as positive control and cell viability (%) was measured using MTT assay. Each value indicates mean ± SD for three independent experiments. Statistically significant differences are indicated by: * (P≤0.05). Panel (C) indicates morphology of the cells after single and combination therapy. The cells were incubated with different concentrations of docetaxel (0, 5, 30 and 50 nM), vinblastine (0, 5, 30 and 40 nM), and their combinations for 48 hrs, and then stained with DAPI. Pictures show the number of cells when treated with docetaxel/vinblastine in comparison with non-treated cells. D) Bar charts represent the number of apoptotic cells which shown as mean±SD calculated from counting a minimum of 3×10² cells in random fields of triplicate wells from three independent experiments. Statistically significant differences are indicated by: (P≤0.05)

Figure 2

Quantification of P-glycoprotein gene/protein levels in H1299 cells incubated with docetaxel, vinblastine and their combinations. Panels (A and B) show the relative mRNA levels of mdr-1 compared to GAPDH as an internal control. The cells were incubated with vinblastine or docetaxel (0, 10, 30 and 40 nM) alone and in combination for 24 hrs, respectively. The results are means ± SD from three independent experiments. Statistically significant differences are indicated by: ** (P ≤0.01); *** (P≤ 0.001). Panel (C) shows the expression of P-gp in various cell lysates analyzed by Western blotting. The cells were treated with vinblastine (30 nM), docetaxel (40 nM) and their combinations for 48 hrs. β-actin protein was used a loading control. The results are representative of three independent experiments. Data were quantified by densitometric analysis using ImageJ software. Panel (D) indicates the densitometric ratio of P-gp/β-actin for each treatment. Statistically significant differences are indicated by: (P≤0.05)

Figure 3

Combination effects of verapamil with chemotherapeutic agents on proliferation, apoptosis induction, and P-glycoprotein expression in H1299 cells. First, the cells were pre-treated with (10 µM) of verapamil and after 1 hr were challenged with vinblastine or docetaxel to measure cell survival and analyze whether drug resistance can be reversed. Verapamil pre-treated cells were incubated with docetaxel (0-100 nM) (A and B) or with vinblastine (0-100 nM) (C and D) for 24 and 48 hrs. The results are expressed as mean±SD values for three independent experiments. Statistically significant differences are indicated by: * (P≤0.05). E) Effects of verapamil on P-gp expression assessed by Western blot analysis in H1299 cells. The cells were harvested for protein isolation after treatment with docetaxel (40 nM), vinblastine (30 nM), combination of verapamil (10 μM) and docetaxel (40 nM) or vinblastine (30 nM) for 48 hrs. β-actin protein was used a loading control. Results shown are from three experiments performed with three different protein extractions, and were quantified by densitometric analysis using ImageJ software. F) Densitometric ratio of P-gp/β-actin for each treatment. Statistically significant differences are indicated by: (P≤0.05)

Figure 4

Morphology of H1299 cells after single and combination treatment to detect apoptotic nuclei. Bar charts represent the number of apoptotic cells which shown as mean ± SD calculated from counting a minimum of 300 cells in random fields of triplicate wells from three independent experiments. Statistically significant differences are indicated by: (P≤0.05)