The Combination of Zerumbone with 5-Fluorouracil for Sensitizing Colorectal Cancer-Associated Fibroblasts to Treatment

Abstract

The present study aimed to evaluate the synergic effects of combination therapy on 5-fluorouracil (5-FU) resistance-cancer-associated fibroblasts (CAFs) to treatment. Chemotherapy resistance is an important challenge in colorectal cancer (CRC) eradication attention to the tumor microenvironment (TME) is very important. CAFs in the TME play an essential role in cancer chemoresistance and relapse. Additionally, many patients with advanced CRC show resistance to 5-FU therapy. Anti-tumorigenic activities of ZER, a chemopreventive compound derived from the rhizomes of the wild ginger, have been demonstrated. Synergistic and potentiating effects of combination therapy, using herbal and chemical drugs, can improve patients' response. At the first, CAFs were isolated from a CRC patient and sorted by fluorescent-activated cell sorting (FACS), then, confirmed by flow cytometry, and immunocytochemistry (ICC). The effect of 5-FU and ZER on the cell viability was investigated by MTT assay in a dose and time-dependent manner, after that, the expression of vimentin, β-catenin, and survivin was quantified. Apoptosis, cell cycle, and invasion were analyzed by flow cytometry and scratch test, respectively. ZER could significantly sensitize CAFs cells to 5-FU. A combination of 5-FU + ZER revealed a marked decrease in the marker of interest in both mRNA and protein levels compared to control groups, including 5-FU, ZER treated, and untreated cells. Functional evaluation of cells in different groups presented significant suppression in migration of CAFs and an apparent increase in cell arrest and apoptosis by 5-FU + ZER treatment.

Figure 1

Isolation and characterization of fibroblastic-like cells from human CRC tumor. (a) Characterization of fibroblasts: primary human fibroblast isolated from tumor was characterized by flow cytometry for α-SMA marker (green). αSMA-positive fibroblastic cells were upregulated in cells after sorting compared to control. (b) The level of α-SMA expression in fibroblastic cells analyzed by ICC, α-SMA positive cells (fibroblastic cells after FACS), and control cells (fibroblastic cells before FACS). (c) FAP and α-SMA mRNA level measured by real-time PCR (relative to before sorting as a control). Mean ± SEM. ^∗∗p < 0.05, ^∗∗p < 0.001compared to control (unsorted control).

Figure 2

The effect of ZER on CAF cells viability. (a) Various concentrations of ZER (0–100 μM) and (b) 5-FU (0–300 μM), and (c) combination of both for 24 h, 48 h, and 72 h. (d) Mix of three treatments on the proliferation of CAFs were determined using MTT assay. Inhibitory concentration (IC50) of ZER, and ZER + 5-FU (22 μM and 50 μM, respectively). No inhibition of growth was seen by 5-FU alone. Data are reported as the mean ± SME (=3). ^∗∗∗p < 0.0001, ≠p < 0.05 compared to control (untreated) and ZER treated, respectively. Each data point is presented as mean ± SED (n = 3).

Figure 3

The effect of ZER on colony-forming of CAF cells. Cells were treated with ZER (22 μM), 5-FU (300 μM), and ZER + 5-FU (22 μM and 50 μM, respectively) for 48 h. Significant inhibition was found in ZER treated group. A more inhibitory effect was observed by the ZER + 5-FU treatment. Each data point is presented as mean ± SED (n = 3). ^∗∗p < 0.01, ^∗∗∗p < 0.001 compared to control (untreated), ^#p < 0.05 compared to ZER treated group.

Figure 4

The effect of ZER on apoptosis of CAF cells. (a) CAF cells were stained with AnnexinV-PI after treatment with 22 μM of ZER for 48 h and 50 μM of 5-FU for 72 h, and ZER + 5-FU for 72 h. (b) The ZER and ZER + 5-FU could promote cell apoptosis vs. the untreated and 5-FU treated cells, respectively (^∗∗∗p < 0.001^∗∗∗P < 0.001) and (^∗∗p < 0.01^∗∗P < 0.01) (n = 1).

Figure 5

The effect of ZER on the cell cycle of CAF cells. (a) CAF cells were incubated with propylene sodium after treatment of CAF cells with 22 μM of ZER for 48 h and 50 μM of 5-FU for 72 h. (b) CAF cell cycle was arrested in S phase markedly in ZER, and ZER + 5-FU treated cells vs. the untreated and 5-FU treated, respectively (^∗∗p < 0.01^∗∗P < 0.01) (^∗p < 0.05^∗P < 0.05). (c) The ZER and ZER + 5-FU could increase apoptotic cells in sub-G1 vs. the untreated and 5-FU treated cells, respectively (^∗∗p < 0.01^∗∗P < 0.01) (^∗p < 0.05^∗P < 0.05) (n = 1).

Figure 6

The effect of ZER on the expression of β. Catenin, survivin, and vimentin by real-time-PCR and Western blotting. CAF cells were treated with 22 μM ZER for 48 h, 50 μM 5-FU for 72 h, and 5-FU + ZER for 72 h. (a) ZER and ZER + 5-FU could markedly inhibit all three biomarkers in mRNA and protein level compared to untreated and ZER-treated groups, respectively (^∗∗p < 0.01^∗∗P < 0.01 vs untreated) (^∗p < 0.05^∗P < 0.05 vs ZER-treated) in mRNA level, and (^∗∗∗p < 0.001 vs untreated), and (^∗p < 0.05^∗P < 0.05, ^∗∗∗p < 0.001^∗∗∗P < 0.001 vs ZER treated). Each data point is presented as mean ± SED (n = 3).

Figure 7

The effect of ZER and 5-FU on CAFs cell migration. (a) CAFs were treated with 5-FU, ZER, and ZER + 5-FU at different time points. (b) Semiquantitative analysis of migration assay, significant reduction of cell migration in ZER, and combination of ZER + 5-FU.