Table 1.
Clinical data for colorectal cancer study participants.
Table 2.
Demographic characteristics of individuals for serum analysis.
Table 3.
Serum concentration of citrulline and arginine in normal volunteers and patients with colorectal cancer (mean ± SD).
Figure 1.
Chromatogram of HPLC for L-citrulline and L-arginine in colorectal tissues.
The upper panel (a) shows the result from paired adjacent normal sigmoid flexure tissue in a patient with sigmoid colon cancer. The lower panel (b) shows the result from sigmoid flexure cancer tissue in the same patient. The individual marked peaks (1) and (2) represent L-citrulline and L-arginine respectively.
Figure 2.
Concentration of Arg and Cit in colorectal cancer tissues and matched normal colon tissues from 30 colorectal cancer patients.
Concentrations of both Arg and Cit were significantly higher in colorectal cancer tissues compared with paired adjacent normal colon tissues (P<0.05 and P<0.01 respectively). The detailed concentrations and statistical analyses are shown in Table 4.
Table 4.
The concentration of citrulline and arginine in colorectal cancer tissues and paired adjacent normal colon tissues (mean ± SD).
Figure 3.
Overexpression of CAT mRNA in tumor relative to normal colon.
The expression of CAT mRNA in colorectal cancer tissues was measured by qRT-PCR, and overexpression was defined as at least 3-fold higher expression than that in normal colon tissue. The figure shows the percentage of samples with overexpression (>3 fold) of individual arginine transporter genes among122 CRC tissue samples. The CAT-1 gene was overexpressed in 86 of 122 (70.5%) CRC tissues.
Figure 4.
CAT-1 protein expression in colorectal cancer tissues by tissue microarray.
The CRC tissue microarray (TMA) was stained with CAT-1 antibody and visualized using a DAB staining kit. The density of CAT-1 expression in the normal colon, well differentiated CRC, and poorly differentiated CRC samples from TMA was compared. The images were taken at 40× magnification.
Figure 5.
CAT-1 RNAi suppressed the cell growth of colorectal cancer cells.
The colon cancer cell line HCT-116 was cultured in vitro in 6-well plates and were transfected with individual siRNAs, followed by analyzing the CAT-1 expression by qRT-PCR (A,B), apoptosis 72 hours after siRNA transfection by flow cytometry (C–E), and cell growth by MTT assay (F). CAT-1 siRNA successfully knocked down approximately 80% of CAT-1 expression (B). Compared with no treatment (C) and control siRNA (D), CAT-1 siRNA induced apoptosis by up to 16.37% (E). CAT-1 siRNA (triangle) significantly reduced cell viability of HCT116 colon cancer cells compared with no treatment and control siRNA transfection (F). The results were reproducible for three independent experiments. ** indicates P<0.01.