Curcumin chemosensitizes 5-fluorouracil resistant MMR-deficient human colon cancer cells in high density cultures

Abstract

Objective: Treatment of colorectal cancer (CRC) remains a clinical challenge, as more than 15% of patients are resistant to 5-Fluorouracil (5-FU)-based chemotherapeutic regimens, and tumor recurrence rates can be as high as 50-60%. Cancer stem cells (CSC) are capable of surviving conventional chemotherapies that permits regeneration of original tumors. Therefore, we investigated the effectiveness of 5-FU and plant polyphenol (curcumin) in context of DNA mismatch repair (MMR) status and CSC activity in 3D cultures of CRC cells.

Methods: High density 3D cultures of CRC cell lines HCT116, HCT116+ch3 (complemented with chromosome 3) and their corresponding isogenic 5-FU-chemo-resistant derivative clones (HCT116R, HCT116+ch3R) were treated with 5-FU either without or with curcumin in time- and dose-dependent assays.

Results: Pre-treatment with curcumin significantly enhanced the effect of 5-FU on HCT116R and HCR116+ch3R cells, in contrast to 5-FU alone as evidenced by increased disintegration of colonospheres, enhanced apoptosis and by inhibiting their growth. Curcumin and/or 5-FU strongly affected MMR-deficient CRC cells in high density cultures, however MMR-proficient CRC cells were more sensitive. These effects of curcumin in enhancing chemosensitivity to 5-FU were further supported by its ability to effectively suppress CSC pools as evidenced by decreased number of CSC marker positive cells, highlighting the suitability of this 3D culture model for evaluating CSC marker expression in a close to vivo setting.

Conclusion: Our results illustrate novel and previously unrecognized effects of curcumin in enhancing chemosensitization to 5-FU-based chemotherapy on DNA MMR-deficient and their chemo-resistant counterparts by targeting the CSC sub-population. (246 words in abstract).

Figure 1. Cell viability is reduced by 5-FU, curcumin and the combination treatment in HCT116, HCT116+ch3 and the corresponding 5-FU resistant cell lines.

HCT116, HCT116+ch3, HCT116R and HCT116+ch3R cell lines were treated with different concentrations of 5-FU (0, 1, 5, 10 and 20 µM) alone (A) for 24 hours, different concentrations of curcumin (0, 1, 5, 10 and 20 µM) alone (B) for 24 hours, or were pre-treated with curcumin 5 µM for 4 h, and then exposed to different concentrations of 5-FU (0, 0.1, 1, 2, 4) for 24 hours (C). Cell viability was measured with the MTT method and IC₅₀ determined at 50% growth inhibition. 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. Significant values are marked with (*).

Figure 2. Curcumin inhibits colonosphere formation in HCT116, HCT116+ch3 and their respective 5-FU-chemoresistant cell lines by heightening the chemosensitivity to 5-FU treatment in high density cultures.

High density cultures of HCT116 (A) or HCT116R (B) cells were either left untreated or were treated with 5-FU (5 µM), curcumin (20 µM), or 5-FU/curcumin in combination (0.1/5 µM). Cultures were evaluated after 1, 3, 7, and 10 days, and pictures of the native cultures taken. Pictures are representative of three individual experiments. Colonosphere size was measured and results presented are mean values with standard deviations from three independent experiments. C: High density cultures of HCT116, HCT116+ch3 and their respective 5-FU-chemoresistant cells were either left untreated or were treated with 5-FU (5 µM), curcumin (20 µM), or 5-FU/curcumin in combination (0.1/5 µM). Cultures were evaluated after 10 days, colonosphere size was measured and results presented are mean values with standard deviations from three independent experiments. Significant values are marked with (*).

Figure 3. Cytotoxicity of 5-FU, curcumin and the combination treatment on colon cancer cells in high density cultures.

High density cultures of HCT116, HCT116+ch3 (A), or HCT116R and HCT116+ch3R (B) were either left untreated or were treated with 5-FU (5 µM), curcumin (20 µM), or 5-FU/curcumin in combination (0.1/5 µM). Cultures were evaluated after 1, 3, 7, and 10 days, and stained with Hoechst 33258 (DAPI) to reveal apoptotic changes of the cell nuclei. Pictures are representative of three individual experiments.

Figure 4. Ultrastructural evaluation of cytotoxicity of 5-FU, curcumin and the combinational treatment on HCT116 and HCT116+ch3 cells in high density cultures.

A: High density cultures of HCT116 and HCT116+ch3 were either left untreated or were treated with 5-FU (5 µM), curcumin (20 µM), or 5-FU/curcumin in combination (0.1/5 µM). Cultures were evaluated after 1, 3, 7, and 10 days, and evaluated ultrastructurally with a transmission electron microscope. At the earliest time point when apoptosis (arrows) was first detected, images are highlighted in red boxes. Micrographs shown are representative of three individual experiments. Magnification: x5000, bar = 1 µm. B: Mitochondrial changes (MC) and apoptosis were quantified by counting 100 cells with morphological features of apoptotic cell death from 25 different microscopic fields and results presented are mean values with standard deviations from three independent experiments. Significant values are marked with (*).

Figure 5. Ultrastructural evaluation of cytotoxicity of 5-FU, curcumin and the combinational treatment on HCT116 5-FU-chemoresistant and HCT116+ch3 5-FU-chemoresistant cell lines in high density cultures.

A: High density cultures of HCT116R and HCT116+ch3R were either left untreated or were treated with 5-FU (5 µM), curcumin (20 µM), or 5-FU/curcumin in combination (0.1/5 µM). Cultures were evaluated after 1, 3, 7, and 10 days, and evaluated ultrastructurally with an electron microscope. At the earliest time point when apoptosis (arrows) was first detected images are highlighted in red boxes. Micrographs shown are representative of three individual experiments. Magnification: x5000, bar = 1 µm. B: Mitochondrial changes (MC) and apoptosis were quantified by counting 100 cells with morphological features of apoptotic cell death from 25 different microscopic fields and results presented are mean values with standard deviations from three independent experiments. Significant values are marked with (*).

Figure 6. Effect of curcumin on 5-FU-induced apoptotic signaling in HCT116, HCT116+ch3 and their respective 5-FU-chemoresistant cell lines in high density cultures.

High density cultures of HCT116 and HCT116+ch3 (left lanel) and of HCT116R and HCT116+ch3R (right panel) cells were either left untreated or were treated with 5-FU (5 µM), curcumin (20 µM), or 5-FU/curcumin in combination (0.1/5 µM). Cultures were evaluated after 3 days and whole cell lysates prepared and analyzed by western blotting for cleavage of PARP. Western blots shown are representative of three independent experiments. The housekeeping protein β-actin served as a positive loading control in all experiments.

Figure 7. Colon cancer stem cell marker expression in high density cultures as shown by western blotting evaluation.

High density cultures of HCT116 and HCT116+ch3 (left lanel, A, B, C) and of HCT116R and HCT116+ch3R (right panel, A, B, C) cells were either left untreated or were treated with 5-FU (5 µM), curcumin (20 µM), or 5-FU/curcumin in combination (0.1/5 µM). Cultures were evaluated after 3 days and whole cell lysates prepared and analyzed by western blotting and quantitative densitometry for expression of CD133, CD44 and ALDH1. Western blots shown are representative of three independent experiments. The housekeeping protein β-actin served as a positive loading control in all experiments. Significant values are marked with (*).