Down-regulation of cIAP2 enhances 5-FU sensitivity through the apoptotic pathway in human colon cancer cells

Abstract

Currently 5-fluorouracil (5-FU) plays a central role in the chemotherapeutic regimens for colorectal cancers and thus it is important to understand the mechanisms that determine 5-FU sensitivity. The expression profiles of human colon cancer cell line DLD-1, its 5-FU-resistant subclone DLD-1/FU and a further 21 types of colon cancer cell lines were compared to identify the novel genes defining the sensitivity to 5-FU and to estimate which population of genes is responsible for 5-FU sensitivity. In the hierarchical clustering, DLD-1 and DLD-1/FU were most closely clustered despite over 100 times difference in their 50% inhibitory concentration of 5-FU. In DLD-1/FU, the population of genes differentially expressed compared to DLD-1 was limited to 3.3%, although it ranged from 4.8% to 24.0% in the other 21 cell lines, thus indicating that the difference of 5-FU sensitivity was defined by a limited number of genes. Next, the role of the cellular inhibitor of apoptosis 2 (cIAP2) gene, which was up-regulated in DLD-1/FU, was investigated for 5-FU resistance using RNA interference. The down-regulation of cIAP2 efficiently enhanced 5-FU sensitivity, the activation of caspase 3/7 and apoptosis under exposure to 5-FU. The immunohistochemistry of cIAP2 in cancer and corresponding normal tissues from colorectal cancer patients in stage III revealed that cIAP2 was more frequently expressed in cancer tissues than in normal tissues, and cIAP2-positive patients had a trend toward early recurrence after fluorouracil-based chemotherapy. Although the association between drug sensitivity and the IAP family in colorectal cancer has not yet been discussed, cIAP2 may therefore play an important role as a target therapy in colorectal cancer.

Figure 1

(A) Proliferation assays of DLD‐1 and DLD‐1/FU cells. The cell numbers were determined on the indicated days using trypan blue counting. The data represent the means ± SD of triplicate cultures. (B) Cytotoxity of 5‐FU and 50% inhibitory concentration in DLD‐1 and DLD‐1/FU cells. The MTS assay was performed 72 h after treatment with 5‐FU. The data represent the means ± SD of three independent experiments.

Figure 2

(A) A dendrogram and its image plot of the hierarchical clustering analysis of 23 colon cancer cell lines including DLD‐1 and DLD‐1/FU. A cohort of 289 genes out of 13 472 genes, the expression ratios of which varied by SDs of >1.25, were filtered using program Cluster 3.0. (B) The 50% inhibitory concentration of 5‐FU in 23 colon cancer cell lines ranged from 0.00998 to 55.1 µg/mL. The data represent the means of two or three independent experiments. (C) Percentages of differentially expressed genes compared to DLD‐1. The population of genes with more than a two‐fold change in comparison to DLD‐1 ranged from 3.3% to 24.0%.

Figure 3

Down‐regulation of cellular inhibitor of apoptosis 2 (cIAP2) by small interfering RNA (siRNA). The messenger RNA (mRNA) and protein levels of cIAP2 were assessed by (A) real‐time reverse transcription – polymerase chain reaction or (B) Western blotting in DLD‐1/FU, HCT‐8 and HT‐29 under transfection of cIAP2 siRNA. As controls, untransfected cells (control) and cells transfected with control siRNA (ctr siRNA) were used. mRNA were isolated 48 or 120 h after transfection (Fig. 3a). Cell lysates were collected 72 h after transfection (Fig. 3b). (C) Effect of the down‐regulation of cIAP2 on 5‐FU sensitivity in human colon cancer cell lines. In vitro cytotoxicity assay was performed using MTS assay. At 48 h after transfection with cIAP2 siRNA or control siRNA, 5‐FU was added at various concentrations. 72 h after treatment with 5‐FU, MTS assay was performed to determine the 50% inhibitory concentration. The results represent the means ± SD of three independent experiments.

Figure 4

(A) Down‐regulation of cellular inhibitor of apoptosis 2 (cIAP2) induced caspase 3/7 activation. At 48 h after cIAP2 small interfering RNA (siRNA) transfection, cells were exposed to distilled water (DW) or 5‐fluorouracil (5‐FU) for 48 h and measured for caspase 3/7 activity. As controls, the cells transfected with control siRNA (ctr siRNA) were used. The results represent the ratios to caspase 3/7 activity of untransfected cells with common culture. *P < 0.05 versus cells treated with ctr siRNA + DW. **P < 0.05 versus cells treated with each of the other treatments. (B) Incidence of apoptosis in DLD‐1/FU revealed by flow cytometric analysis stained with annexin V‐fluorescein isothiocyanate (V‐FITC) and counterstained with propidium iodide (PI). In cIAP2 siRNA‐transfected cells with exposure to 5‐FU, the percentage of annexin V‐positive/PI‐negative cells (early apoptotic cells) shown in the right lower quadrant was high in comparison to the control siRNA‐transfected cells. (C) The histogram represents the increase of annexin V‐positive fraction in cIAP2 siRNA‐transfected cells with exposure to 5‐FU. A representative assay out of three independent assays was provided.

Figure 5

(A) Cellular inhibitor of apoptosis 2 (cIAP2) expression in human colorectal cancer and the corresponding normal tissues by immunohistochemistry. cIAP2 was more frequently expressed in cancer tissues than in normal tissues. (B) The incidence of cancer recurrence after curative operations in cIAP2‐positive and cIAP2‐negative patients. (C) The time to recurrence in cIAP2‐positive and cIAP2‐negative patients. (D–G) Representative image of immunohistochemical staining for cIAP2 in human colorectal cancer and normal tissues (D, cancer tissue/cIAP2‐positive; E, cancer tissue/cIAP2‐negative; F, normal tissue/cIAP2‐positive; G, normal tissue/cIAP2‐negative; original magnification: ×100).