Curcumin enhances the effect of chemotherapy against colorectal cancer cells by inhibition of NF-κB and Src protein kinase signaling pathways

Abstract

Objective: Development of treatment resistance and adverse toxicity associated with classical chemotherapeutic agents highlights the need for safer and effective therapeutic approaches. Herein, we examined the effectiveness of a combination treatment regimen of 5-fluorouracil (5-FU) and curcumin in colorectal cancer (CRC) cells.

Methods: Wild type HCT116 cells and HCT116+ch3 cells (complemented with chromosome 3) were treated with curcumin and 5-FU in a time- and dose-dependent manner and evaluated by cell proliferation assays, DAPI staining, transmission electron microscopy, cell cycle analysis and immunoblotting for key signaling proteins.

Results: The individual IC50 of curcumin and 5-FU were approximately 20 µM and 5 µM in HCT116 cells and 5 µM and 1 µM in HCT116+ch3 cells, respectively (p<0.05). Pretreatment with curcumin significantly reduced survival in both cells; HCT116+ch3 cells were considerably more sensitive to treatment with curcumin and/or 5-FU than wild-type HCT116 cells. The IC50 values for combination treatment were approximately 5 µM and 1 µM in HCT116 and 5 µM and 0.1 µM in HCT116+ch3, respectively (p<0.05). Curcumin induced apoptosis in both cells by inducing mitochondrial degeneration and cytochrome c release. Cell cycle analysis revealed that the anti-proliferative effect of curcumin and/or 5-FU was preceded by accumulation of CRC cells in the S cell cycle phase and induction of apoptosis. Curcumin potentiated 5-FU-induced expression or cleavage of pro-apoptotic proteins (caspase-8, -9, -3, PARP and Bax), and down-regulated anti-apoptotic (Bcl-xL) and proliferative (cyclin D1) proteins. Although 5-FU activated NF-κB/PI-3K/Src pathway in CRC cells, this was down-regulated by curcumin treatment through inhibition of IκBα kinase activation and IκBα phosphorylation.

Conclusions: Combining curcumin with conventional chemotherapeutic agents such as 5-FU could provide more effective treatment strategies against chemoresistant colon cancer cells. The mechanisms involved may be mediated via NF-κB/PI-3K/Src pathways and NF-κB regulated gene products.

Figure 1. Effect of curcumin and/or 5-FU on cell viability and proliferation of HCT116 and HCT116+ch3 colon cancer cells.

A: HCT116 cells were treated with different concentrations of curcumin or 5-FU (0, 1, 5, 10, 20, 40 and 80 µM) for 24 h and cell viability was measured using the MTT method. Concentrations of curcumin and 5-FU resulting in 50% growth inhibition were indicated as individual IC₅₀ values. B: HCT116 cells were pretreated with curcumin (5 µM for 4 h), then exposed to 5-FU in different concentrations (0, 0.1, 1, 2, 3, 4 and 5 µM) for 24 h and evaluated by MTT assay. IC₅₀ for 5-FU in combination treatment was determined at 50% growth inhibition of the HCT116 cells. The same experiments shown in (A) and (B) were performed on HCT116+ch3 cells. IC₅₀ values for single (C) and combination treatment (D) were calculated on the basis of MTT measurements. The results are provided as mean values with standard deviations from at least three independent experiments. Values were compared to the control and statistically significant values with p<0.05.

Figure 2. Effect of curcumin and/or 5-FU on apoptosis in HCT116 and HCT116+ch3 colon cancer cells.

HCT116 and HCT116+ch3 cells were treated with different concentrations of curcumin or 5-FU (0, 1, 5, 10 and 20 µM) or a combination of curcumin (5 µM) and 5-FU (0.1, 1, 2 and 3 µM) for 24 h. Monolayer cultures were stained with Hoechst 33258 (DAPI) to reveal apoptotic changes in the cell nuclei.

Figure 3. Ultrastructural evaluation of mitochondrial and apoptotic changes after treatment with curcumin and/or 5-FU in HCT116 and HCT116+ch3 colon cancer cells.

HCT116 cells were treated with curcumin (20 µM), 5-FU (5 µM) or a combination of both (5 µM curcumin and 1 µM 5-FU) for 12, 24, 36, 48, 60 and 72 h. Using a different approach HCT116+ch3 cells were treated with a combination of 5 µM curcumin and 0.1 µM 5-FU for 12, 24, 36, 48, 60 and 72 h. Ultrathin sections were prepared and evaluated by transmission electron microscopy. Micrographs shown are representative of all the cultures evaluated. At the earliest time point when apoptosis was first detected images are highlighted arrows. Mitochondrial changes (arrowheads) are shown. Magnification: x5000, bar = 1 µm.

Figure 4. Quantification of mitochondrial and apoptotic changes after treatment with curcumin and/or 5-FU in HCT116 and HCT116+ch3 colon cancer cells.

To quantify the ultrastructural findings, HCT116 (A) and HCT116+ch3 (B) cultures treated as described in Fig. 3 were examined for apoptotic and mitochondrial changes (MC) by counting 100 cells from 20 microscopic fields. The examination was performed in triplicate and the results are provided as mean values with standard deviations SD (p<0.05) from three independent experiments.

Figure 5. Effect of curcumin and/or 5-FU on the cell cycle of HCT116 and HCT116+ch3 colon cancer cells.

HCT116 cells were treated with 20 µM curcumin or 5 µM 5-FU or a combination of 5 µM curcumin and 1 µM 5-FU for 12 and 24 h (A). HCT116+ch3 cells were treated with 5 µM curcumin or 1 µM 5-FU or a combination of 5 µM curcumin and 0.1 µM 5-FU for 12 and 24 h (B). Cell cycle analysis was performed by flow cytometry. These studies were performed in triplicate and the results presented are mean value with standard deviations from three independent experiments. Values are given as mean ± SD (p<0.05).

Figure 6. Effect of curcumin and/or 5-FU on apoptotic signaling in HCT116 and HCT116+ch3 colon cancer cells.

HCT116 cells were treated with 20 µM curcumin or 5 µM 5-FU or a combination of 5 µM curcumin (4 h pretreatment) and 1 µM 5-FU for 24 h. HCT116+ch3 cells were treated with 5 µM curcumin or 1 µM 5-FU or a combination of 5 µM curcumin (4 h pretreatment) and 0.1 µM 5-FU for 24 h. Whole cell lysates were prepared and analyzed by western blotting for A: expression or cleavage of pro-apoptotic proteins caspase-8, caspase-9, caspase-3, PARP and Bax, and of anti-apoptotic protein BCL-xL B: expression of cyclin D1. The housekeeping protein β-actin served as a positive loading control in all experiments.

Figure 7. Effect of curcumin and/or 5-FU on mitochondrial damage and cytochrome c release in HCT116 and HCT116+ch3 colon cancer cells.

HCT116 cells were treated with 20 µM curcumin or 5 µM 5-FU or a combination of 5 µM curcumin (4 h pretreatment) and 1 µM 5-FU for 24 h. HCT116+ch3 cells were treated with 5 µM curcumin or 1 µM 5-FU or a combination of 5 µM curcumin (4 h pretreatment) and 0.1 µM 5-FU for 24 h. Mitochondrial and cytoplasmic cell fractions were prepared and analyzed by western blotting using antibodies against cytochrome c. The housekeeping protein β-actin served as a loading control.

Figure 8. Effect of curcumin and/or 5-FU on NF-κB and PI-3K/Src activation in HCT116 and HCT116+ch3 colon cancer cells.

HCT116 and HCT116+ch3 cells were either treated with different concentrations of 5-FU (0, 2, 5, 10, 20, 40 µM) alone for 1 h or were pretreated with different concentrations of curcumin (0, 2, 5, 10, 20, 40 µM) for 1 h and then exposed to 1 µM (HCT116) or 0.1 µM (HCT116+ch3) 5-FU for 1 h. A: After preparation of nuclear extracts western blotting was performed with antibodies against NF-κB and PARP as a loading control. B: Cytoplasmic fractions were subsequently examined by western blotting for expression of PI-3K (lane I), Src (lane II) and β-actin (lane III) (loading control).

Figure 9. Effect of 5-FU and/or curcumin or PI-3K inhibitor wortmannin on activation of IκBα kinase (IKK) in HCT116 and HCT116+ch3 colon cancer cells.

A: HCT116 cells were treated with 5-FU (5 µM) for 0, 5, 10, 20, 40, or 60 minutes or were pretreated with curcumin (5 µM) or wortmannin (10 nM) for 1 h and then co-treated with 1 µM 5-FU for 0, 5, 10, 20, 40, or 60 minutes. B: HCT116+ch3 cells were treated with 5-FU (1 µM) for 0, 5, 10, 20, 40, or 60 minutes or were pretreated with curcumin (5 µM) or wortmannin (10 nM) for 1 h and then co-treated with 0.1 µM 5-FU for 0, 5, 10, 20, 40, or 60 minutes. Cells were lysed and immune complex kinase assays were performed as described in Materials and Methods. Equal amounts of total protein (500 ng protein per lane) were separated by SDS-PAGE under reducing conditions and then analyzed by immunoblotting using antibodies against phosphospecific IκBα (lane I), IKK-α (lane II), and IKK-β (lane III).

Figure 10. Schematic diagram illustrating the signaling pathways involved in the development of chemotherapeutic treatment resistance to 5-FU and chemosensitization by curcumin in HCT116 and HCT116+ch3 colon cancer cells.