The human CRC cell line HCT-8 and 5-fluorouracil (5-Fu)-resistant HCT-8 cells (HCT-8/5-Fu) were purchased from KeyGen Biotech Co. Ltd. (Nanjing, Jiangsu, China). HCT-8 cells were cultured in RPMI-1640 medium supplemented with 10% inactivated fetal calf serum. HCT-8/5-Fu cells were cultured in RMPI-1640 medium supplemented with 15 mg/l 5-Fu.

Anti-TrpC5 (ACC-020) was purchased from Alomone Labs (Jerusalem, Israel) and anti-glucose transporter 1 (GLUT1) (ab115730) and anti-c-Myc (ab32) were purchased from Abcam Biotechnology (Cambridge, MA, USA), and anti-histone (AH433) and anti-β-actin (AA128) were from Beyotime Biotechnology (Nantong, Jiangsu, China). The secondary antibodies, a goat anti-rabbit IgG (A0208) and a goat anti-mouse IgG (A0216), were purchased from Beyotime Biotechnology. The Nuclear and Cytoplasmic Protein Extraction kit (P0027) was purchased from Beyotime Biotechnology. TRIzol (10296–010) and Lipofectamine 2000 (11668–019) were purchased from Invitrogen (Camarillo, CA, USA). TrpC5-shRNA (sc-42670) was purchased from Santa Cruz Biotechnology (Dallas, TX, USA). Dimethyl sulfoxide (DMSO), 3-BrPA, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) and XAV939 were purchased from Sigma Chemical Co. (St. Louis, MO, USA). 5-Fu was purchased from Jinyao Amino Acid Co., Ltd. (Tianjin, China).

When the cells reached 50–70% confluency, the HCT-8/5-Fu cells were treated with a TrpC5-shRNA (indicated as HCT-8/5-Fu/RNAi) (scrambled siRNA was used as a control and was indicated as HCT-8/5-Fu/scrambled). XAV939 (an inhibitor of the canonical Wnt/β-catenin signaling pathway) was used in the present study. HCT-8/5-Fu cells were treated with XAV939 (10 µM, overnight) (indicated as HCT-8/5-Fu/XAV939) (DMSO was used as a control and was indicated as HCT-8/5-Fu/DMSO). Real-time PCR and western blotting were used to determine the expression of TrpC5, GLUT1 and c-Myc.

The whole-cell protein lysate was obtained using RIPA buffer containing 1 mM phenylmethylsulphonyl fluoride (PMSF). The nuclear proteins were isolated using a Nuclear and Cytoplasmic Protein Extraction kit. The same quantity of total proteins was electrophoresed on a 10% polyacrylamide gel containing 0.1% SDS. The resolved proteins were semi-dry-transferred to a polyvinylidene difluoride (PVDF) membrane. The primary antibodies, anti-TrpC5 (1:500), anti-GLUT1 (1:1,000) and anti-c-Myc (1:500), were used to detect the expression of the proteins of interest. β-actin and histone were the internal references. The antigen-antibody complexes were visualized by an enhanced chemiluminescent reaction. The protein bands were analyzed using ImageJ software (NIH, Bethesda, MD, USA).

Total RNA was extracted from the cells using TRIzol. The procedure for the real-time PCR was previously reported (20). The primer pairs are listed in Table I.

Briefly, 10⁴ cells (200 µl) were seeded into 96-well plates. Twelve hours later, the cells were treated with medium containing different concentrations of 5-Fu for 48 h. Then, the medium in each well was replaced with 200 µl of fresh RPMI-1640 containing 5 mg/ml MTT for 4 h. DMSO (150 µl) was added to each well and then, the absorbance was determined at 490 nm.

We retrospectively screened advanced CRC patients with unresectable metastasis who underwent a biopsy and/or surgery for a primary lesion at the Affiliated Hospital of Jiangnan University (The Fourth People's Hospital of Wuxi) from January 2009 to December 2014. Patients who postoperatively received 5-Fu-based first-line systematic chemotherapy were enrolled in the present study. The following were the exclusion criteria: i) patients receiving a localized treatment to the target lesions, such as radiotherapy or radiofrequency ablation; ii) patients receiving preoperative cytotoxic treatment, such as radiation or chemotherapy; iii) patients with a diameter of the maximum target lesion was <1 cm by CT-scan; and iv) patients receiving postoperative systematic chemotherapy for <2 cycles. Tumor assessment was performed after every 2 cycles of chemotherapy according to the Response Evaluation Criteria in Solid Tumors 1.1 (RECIST 1.1) criteria (22), and the assessment was classified as a complete response (CR), a partial response (PR), stable disease (SD) and progressive disease (PD). Ethical permission was obtained from the Ethics Committee at the Affiliated Hospital of Jiangnan University (The Fourth People's Hospital of Wuxi).

CRC tissue slides were de-paraffinized with xylene and rehydrated through a graded alcohol series. After incubation with 10% bovine serum albumin, the slides were then incubated with the primary antibodies [TrpC5 (1:2,000) and GLUT1 (1:100)] overnight at 4°C in a humidified chamber and were subsequently incubated with the secondary antibodies. The results of immunostaining were assessed by two pathologists, respectively. Five visual fields were selected from each slide. The results were judged according to the German semi-quantitative scoring system (23) (no staining, 0; weak staining, 1; moderate staining, 2; and strong staining, 3), and the extent of stained cells (0%=0; 1–24%=1; 25–49%=2; 50–74%=3; and 75–100%=4). The final immunoreactive score was determined by multiplying the intensity score with the extent of score of the stained cells, ranging from 0 (the minimum score) to 12 (the maximum score) and was defined as follows: ± (0–3), + (3.1–6), ++ (6.1–9) and +++ (9.1–12). Each grade of TrpC5 and GLUT1 was from the same sample. The expression levels were categorized as low (± and +) or high (++ and +++).

The results are presented as the mean ± standard error. Statistical significance was determined by a Student's t-test and a Pearson's Chi-squared test as applicable. A value of p<0.05 was considered to indicate a statistically significant result.

In the present study, GLUT1 expression was examined to represent the glycolytic level. 5-Fu-resistant human CRC cells (HCT-8/5-Fu) and HCT-8 parental cell lines were used in the present study. An MTT assay revealed that the half maximal inhibitory concentration of 5-Fu (5-Fu IC₅₀) for the HCT-8/5-Fu and HCT-8 cells was 86.0 mg/l [95% confidence interval (CI), 72.2–102.5 mg/l] and 1.9 mg/l (95% CI, 1.8–2.1 mg/l) (p<0.05), respectively (Fig. 1A). Real-time PCR analysis showed that the expression of both TrpC5 and GLUT1 at the mRNA level in the HCT-8/5-Fu cells was higher than the levels in the HCT-8 cells (Fig. 1B). This finding was validated by western blotting. The protein expression of TrpC5 and GLUT1 was higher in the HCT-8/5-Fu cells, whereas only a low level was detected in the HCT-8 parental cell line (Fig. 1C).

The possible role of TrpC5 in controlling GLUT1 expression was explored. The real-time PCR analysis demonstrated that, when the HCT-8/5-Fu cells were treated with TrpC5-siRNA, the TrpC5 and GLUT1 mRNA levels were both reduced compared with the cells treated with the scrambled siRNA (Fig. 2A). Furthermore, a western blot assay gave similar results. The TrpC5 and GLUT1 protein expression levels in the HCT-8/5-Fu/RNAi group were significantly lower than these levels in the HCT-8/5-Fu/scrambled cells (Fig. 2B).

In our previously study, upregulated expression of TrpC5 in human CRC cells activated the Wnt/β-catenin signaling pathway (20), and the latter was demonstrated to regulate GLUT1 expression through its target gene c-Myc (24,25). We next explored the mechanism of the Wnt/β-catenin signaling pathway and how it is involved in the regulation of GLUT1 by TrpC5. The Wnt/β-catenin signaling pathway was found to be activated in HCT-8/5-Fu cells (20), and in the present study, XAV939 was used to inhibit the Wnt/β-catenin pathway in the HCT-8/5-Fu cells. Western blot analysis showed decreased nuclear c-Myc expression and a markedly decreased GLUT1 expression in the HCT-8/5-Fu cells treated with XAV939 (Fig. 3).

Table II shows the clinical and pathological characteristics of the 72 CRC patients enrolled in the present study. After the first 2 cycles of 5-Fu chemotherapy, 28 patients achieved CR or PR (these were considered responders) and the remaining 44 patients achieved SD or PD (these were considered non-responders). The immunostaining showed that TrpC5 and GLUT1 were mostly localized near the cell surface (Fig. 4). TrpC5 and GLUT1 were found to be highly co-expressed in 21 of the 44 non-responders and in only 4 of the 28 responders. A Pearson's Chi-squared test showed a significant correlation between TrpC5 expression and GLUT1 expression (Table III), and a higher TrpC5/GLUT1 expression was closely related with chemotherapy failure in the advanced CRC patients (Table IV).