Colorectal cancer (CRC) is one of the leading causes of cancer mortality in most courtiers, and globally affects over a million people each year in the developed countries [1]. Distant metastasis through lymphatic or hematogenous dissemination contributes to a poor prognosis for CRC, and the liver is the most frequent site of distant metastasis of CRC [2].

Currently, liver resection remains a standard procedure and the only potentially curative therapy for colorectal liver metastases (CLM). Unfortunately, the initial resection rates are reported to be less than 25% [3], with a high recurrence rate of 70–80% after curative resection [4]. Chemotherapy alone or in addition to local minimally invasive treatment, such as radiofrequency ablation, transarterial chemotherapy, or percutaneous ethanol injection, is the most treatment options in those who are not suitable for resection [5]. However, the underlying mechanism of this aggressive biology of CRC is largely unknown.

Aberrant energy metabolism is a critical hallmark for many types of human tumors [6]. Increasingly evidences have shown that glutamine metabolism plays key roles in tumor growth and invasion, and contributes poor outcomes [7-9]. Glutamine is first catabolyzed to glutamate and than to generate a-ketoglutarate, a tricarboxylic acid (TCA) cycle intermediate. Glutaminolysis supports the viability of cancer cells by supporting ATP production and by the biosynthesis of proteins, lipids, and nucleotides, and suppress oxidative stress through glutathione synthesis [10]. More importantly, oncogenes and tumor suppressors, such as SIRT4, mTORC1, K-RAS and p53, have been implicated in the regulation of glutamine metabolism [11-14]. Accordingly, it positively regulates the mTORC1 pathway by facilitating the uptake of leucine [15], and regulates STAT3 pathway by promoting tyrosine Y705 phosphorylation [9].

Glutamate dehydrogenase (GDH) is an enzyme that plays a pivotal role in glutamine metabolism by converting glutamate to a-ketoglutarate, especially when glucose is insufficient or under hypoxia. Recently, Csibi and coworkers [16] reported that mTORC1 promoted glutaminolysis by activating GDH to facilitate cell proliferation, transformation, and tumor development by repressing SIRT4. Lorin and colleagues [15] found that GDH contributed to autophagy by activating mTORC1 and by limiting the formation of reactive oxygen species in transformed cells. Yang [17] demonstrated that GDH activity is required for glioblastoma cell to survive impairments of glycolysis brought about by glucose deprivation. Moreover, studies found that treatment with epigallocatechin gallate (EGCG), an allosteric inhibitor of GDH, has considerable effect on tumor growth [18,19]. However, the clinical significance and role of GDH expression in colorectal cancer has not yet been investigated.

In the present study, we examined the expression of GDH in CRC and further analyzed the clinical significance of GDH in a cohort of CRC patients. In addition, we explored the potential role of GDH in CRC cell proliferation and motility, which could help to better understand the pathogenesis of CRC and may further provide a novel therapeutic target for the treatment of CRC patients.

Materials

Cell culture, reagents and Lentiviral transduction

The human colon cancer cell lines HCT116, DLD-1, SW480, RKO and LoVo were grown in RPMI-1640 medium supplemented with 10% fetal bovine serum (FBS). The human colon epithelial cell line NCM460 was cultured in MEM medium supplemented with 10% FBS. All cell lines were maintained at 37°C in a humidified atmosphere with 5% CO2.

AG490 and DMSO were obtained from Sigma. The GDH short hairpin RNA (shRNA) was synthesized and cloned into a pLKO.1-TRC vector (Addgene). These vectors were co-transfected into 293 T cells along with the retroviral packaging plasmid. After transfection, the supernatants were harvested and used to infect CRC cells, and the stably transfected cells were selected with puromycin according the manufacturer’s protocol.

Tumor specimens

Twenty freshly frozen CRC samples and corresponding non tumor tissues were obtained from Sun Yat-sen Memorial Hospital of Sun Yat-sen University. In addition, we collected 104 paraffin embedded CRC specimens from our hospital between January 2002 and February 2005. Tumor staging for the specimens was carried out according to the American Joint Committee on Cancer staging criteria. The median follow-up time was 62.5 months (range from 6.7 to 99). The patients’ overall survival (OS) and disease-free survival (DFS) durations were defined as the interval from initial surgery to death and from initial surgery to clinically proven recurrence or metastasis respectively. The study was approved by the Institute Research Ethics Committee at the Sun Yat-sen University, and written informed consent was obtained from each patient.

Immunohistochemical analysis

Sections of paraffin-embedded CRC specimens were prepared and standard immunohistochemical procedures were carried out as previously described [20]. Briefly, samples were deparaffinized and rehydrated, and the endogenous peroxidase activity was quenched. Antigen retrieval was performed, and the sections were blocked with bovine serum albumin and then incubated with anti-GDH antibody (Abcam; 1:200). Sections were washed and then incubated with a biotinylated secondary antibody and visualized with 3,3-diaminobenzidine.The staining results were scored by two pathologists blinded to the clinical data. Staining index was calculated as the product of the staining intensity (0, no staining; 1, weak staining; 2, moderate staining; 3, strong staining) and the proportion of positive cells (0, no positive tumor cells; 1, <10%; 2, 10-35%; 3, 35-70%; 4, >70%). The immunoreactivity score (IRS) was resulted from the multiplication of both parameters. Using this method of assessment, we evaluated GDH expression by determining the IRS, with scores of 0, 1, 2, 3, 4, 6, 8, 9 or 12. The samples were divided into 2 groups as follows: low (IRS = 0–4), and high (IRS ≥6).

RNA extraction and quantitative real-time PCR

Total RNA was extracted using TRIzol reagent. The reverse-transcription PCR (RT-PCR) was performed using transcriptase, and the quantitative real-time PCR (qRT-PCR) was performed in a LightCycler480 System using a SYBR Premix ExTaq kit according to the manufacturer’s instructions. Primers for qRT-PCR are as follows. GDH: Forward, 5′-GGG ATT CTA ACT ACC ACT TGC TCA-3′, Reverse 5′-AAC TCT GCC GTG GGT ACA AT-3′. GAPDH: Forward, 5′-CTC CTC CTG TTC GAC AGT CAG C-3′, Reverse, 5′-CCC AAT ACG ACC AAA TCC GTT-3′. The relative expression levels were calculated by the 2-ΔΔCT method. Each assay was carried out in triplicate.

Western blot analysis

Cell cytosolic protein fractions were prepared using RIPA buffer. According to standard Western blot procedures, briefly, proteins were separated by SDS-PAGE and then transferred to polyvinylidene fluoride (PVDF) membranes. After blocking in 5% nonfat milk, the membranes were incubated with the following primary antibodies: GDH antibody (Abcam), STAT3, pSTAT3 (Tyr705), E-Cadherin, Vimentin, ZEB1 and GAPDH antibody (Cell Signaling Technology) according to the manufacturer’s instructions.

Cell proliferation

Cell proliferation was analysed with the MTT assay. Cells were seeded in 96-well plates at a density of 1,000 cells per well. At 1, 2, 3 and 4 days, the cells were stained with 20 μl of MTT (0.5 mg/ml) for 4 h, and after which the medium was removed, and 100 μl of DMSO was added. The absorbance was measured at 490 nm. The anchorage-independent sphere formation assay was performed by culturing the cells in suspension in serum-free DMEM-F12 supplemented with B27 (Invitrogen), EGF (BD Biosciences, CA, USA), 0.4% bovine serum albumin (Sigma, MO, USA), and insulin (Sigma).

Cell migration and invasion assays

Cell motility was assessed by wound healing assay as previously described [21]. Results were expressed as a migration index: the distance migrated by targeted relative to the distance migrated by control cells. Cell invasion assays were performed using 24-well transwells (8-μm pore size, BD Sciences) coated with matrigel (1 mg/ml, BD Sciences), as previously described [21]. The inserts were stained with 0.2% crystal violet, imaged, and counted under an inverted microscope in six randomly selected fields. All experiments were carried out in triplicate and repeated at least three times.

Statistical analysis

Data are presented as the mean ± SD, and differences between groups were analyzed using Student’s t-test or chi-squared test or fisher exact test. The Kaplan-Meier method and log-rank test were used to estimate survival rates. Cox proportional hazards model was used to calculate univariate and multivariate hazard ratios for the study variables. Statistical analyses were performed with SPSS 16.0 software (Chicago, IL), and P values of < 0.05 were considered statistically significant.