Endocannabinoids and ceramides have demonstrated growth inhibition, cell death induction and pro-apoptotic activity in cancer research. In the present study, we describe the profiles of two major endocannabinoids, ceramides, free fatty acids and relevant metabolic enzymes in 47 pairs of human colorectal cancer tissues and adjacent non-tumor tissues. Among them, anandamide (AEA) and its metabolite, arachidonic acid (AA), were markedly upregulated in cancer tissues particularly in those with lymphatic metastasis. The levels of C16 and C24 ceramides were significantly elevated in the colorectal tumor tissues, while levels of C18 and C20 ceramides showed opposite trends. Levels of two enzymes participating in the biosynthesis and degradation of AEA,
As one of the three most common cancers in the world, colorectal cancer (CRC) occurs with a global incidence exceeding 1.2 million new cases and 600,000 deaths per year. Endoscopic removal of precursor lesions appears to be the only effective treatment strategy for CRC (
As two major endocannabinoids, anandamide (AEA) was first isolated, followed by 2-AG (
Ceramides are vital signaling sphingolipids involved in cell growth arrest, differentiation and apoptosis, and contain two frame structures, a sphinganine base and a fatty acyl with varying lengths of carbon chains (
In order to reveal the expression patterns of endocannabinoids and ceramides in human CRC, we evaluated the endogenic levels of AEA, 2-AG, ceramides and their metabolites via liquid chromatography-tandem mass spectrometry (LC-MS/MS). The expression levels of two cannabinoid receptors and the relevant enzymes, such as NPLD, FAAH, DGL-α, MGL and CerSs, were also analyzed to evaluate the potential effects of these lipids on tumor treatment.
Colorectal cancer samples were obtained from 47 Chinese patients undergoing surgical resection at the Department of Colorectal Surgery, Fujian Medical University Union hospital (Fuzhou, China) during 2011, and were confirmed with TNM stage according to the World Health Organization TNM staging 7th edition by pathological analysis. Non-tumor control tissues were resected from the same patients at the region 5–7 cm above the tumor position. All tissues were divided into several slices for lipid, gene and protein analysis, and were freshly frozen and stored at −80°C immediately after resection. Informed consent was obtained from each patient before surgery. Study of the resected human tissues was approved by the Ethics Committee of Fujian Medical University Union hospital, following clinical registration guidelines in China.
Lipids were extracted from the tissue samples using a previously described method with modifications (
Analysis of enzyme activity was also carried out using a previously described method (
Protein expression levels of the tissue samples were evaluated by western blot analysis. First, the concentration of homogenized tissue protein was measured using the BCA protein assay kit (Pierce, China). Proteins were separated by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), and then transferred to PVDF membranes (Amersham Biosciences, China). Membranes with the proteins were incubated overnight with primary antibodies against the CB1 receptor (1:500), CB2 receptor (1:500; Cayman, China) or β-actin at 4°C, followed by incubation with HRP-conjugated anti-rabbit IgG antibody (1:10,000; Sigma, China) for 1 h at room temperature. Protein bands were visualized using an ECL Plus kit (Amersham Biosciences). mRNA expression levels were measured by RT-qPCR assay. Tissue samples were ground using ice-cold porcelain mortars with TRIzol to extract total RNA. The RNA concentration was measured with a spectrophotometer (Beckman Coulter, USA). Reverse transcription was completed with a ReverTra Ace qPCR RT kit (Toyobo, Japan) following the manufacturer’s instructions. Finally quantitative PCR was performed and analyzed in a 7500 Fast real-time PCR System (Applied Biosystems). The mRNA expression levels were normalized by housekeeping gene 18S as the internal standard.
Primers were as follows: NPLD forward primer (F), 5′-TGGCTGGGACACGCG-3′ and reverse primer (R), 5′-GGGATCCGTGAGGAGGATG-3′; FAAH F, 5′-GCCTCAAGGAATGCTTCAGC-3′ and R, 5′-TGCCCTCATTCAGGCTCAAG-3′; MGL F, 5′-CATGTGGATTCCATGCAGAAAG-3′ and R, 5′-AGGATTGGCAAGAACCAGAGG-3′; DGL-α F, 5′-AGAATGTCACCCTCGGAATGG-3′ and R, 5′-GTGGCTCTCAGCTTGACAAAGG-3′; CB1 F, 5′-AGCCTCTGGATAACAGCATGG-3′ and R, 5′-AATCTTGACCGTGCTCTTGATG-3′; CB2 F, 5′-CTCAGTGACCAGGTCAAGAAGG-3′ and R, 5′-TTTTGCCTCTGACCCAAGG-3′; CerS1 F, 5′-TTTGGCTCCCGCACAATGT-3′ and R, 5′-AAAAGCGAGATA GAGGTCCTCA-3′; CerS2 F, 5′-GCTCTTCCTCATCGTTCGATAC-3′ and R, 5′-GTGTAGCCACGTACAGCTCA-3′; CerS5 F, 5′-GCTGCTCTTCGAGCGATTTAT-3′ and R, 5′-CCTCCGATGGCGAAACCAG-3′; CerS6 F, 5′-TTTGGCTCCCGCACAATGT-3′ and R, 5′-AAAAGCGAGATAGAGGTCCTCA-3′; 18S F, 5′-CAGCCACCCGAGATTGAGCA-3′ and R, 5′-TAGTAGCGACGGGCGGTGTG-3′.
Data of the results are expressed as means ± SEM. Statistical significance was analyzed with one-way variance analysis (ANOVA) followed by the Bonferroni all pairs test. Statistical analysis was completed using GraphPad Prism (GraphPad Software, USA), and the difference was considered statistically significant at p<0.05.
Tumor samples were collected from 47 patients with CRC during surgical removal. Detailed baseline characteristics of the 47 patients are listed in
After detection by LC-MS/MS assay and normalization by the wet weight of each sample, the levels of AEA, 2-AG, ceramides and free fatty acids were analyzed. Regarding the endocanabinoids, the anandamide level was obviously elevated in the CRC tissues when compared with this level in the non-tumor control tissues, and was nearly 2-fold higher in the tissues with metastasis of the lymph nodes (
In order to explain the variation of lipids in human CRC, mRNA expression levels and activities of metabolic enzymes were respectively analyzed by quantitative PCR assay and enzyme activity assay as described above. We first measured the mRnA expression levels and activities of associative metabolic enzymes responsible for AEA and 2-AG. As a main synthetase of AEA, NPLD was 2- to 3-fold overexpressed in the tumor tissues; the enzyme activities of which showed the same upward trend (
Based on the alteration of endocannabinoid levels in the CRC tissues, we further analyzed the protein expression levels of the cannabinoid receptors via western blot analysis in the same tissue samples. CB1 expression was elevated 1.5-fold in the N0 group (0.25±0.01), and 2-fold in the N1+ group (0.31±0.04), which were compared with adjacent non-tumor samples (0.14±0.01) (
Endocannabinoids have been demonstrated to act with multi-function in the gastrointestinal tract, particularly in the colon. D’Argenio
In the present study, we found that the level of AEA was increased in the CRC tissues, particularly in those with lymph node metastasis, while 2-AG levels showed no significant difference. The result of AEA levels was consisting with data reported by Ligresti
AEA elevation in CRC was suggested to be self-protection against further tumor progression. It is known that multiple pharmacological functions of AEA are accomplished through activation of cannabinoid receptors, particular CB1 (
It has been reported that CB1 receptor activation caused sphingomyelin hydrolysis and rapid ceramide production in astrocytes and C6 glioblastoma cells (
We further measured ceramide synthases (CerSs) in human CRC tissues, which are important enzymes for ceramide synthesis. Since specificity of each CerS subtype for ceramides with fatty acyl chain, four abundant CerS subtypes in human colon were evaluated. Unexpectedly, the mRnA levels of all the detected CerSs were sharply increased in the CRC tissues. Upregulated levels of CerS2, CerS5 and CerS6 were explained to synthesize and elevate enough C16 and C24 ceramides to decelerate tumorigenesis. CerS1 showed a contrary upward trend against the reduced levels of its specific C18 ceramide, which may be a defensive mechanism to compensate enough C18 ceramides. Overlapping enzyme activity of CerS2 and CerS6, partly responsible for synthesis of C18 and C20 ceramides, made it difficult to study CerSs and ceramides in a one-to-one relationship. The complex regulatory mechanism of CerSs and ceramides with different carbon chains of fatty acyl still require further in-depth research. Yet, consistent alteration of C16/C18/C24 ceramides in several tumors revealed that they may be potential indicators in tumor monitoring.
In addition to AA, a metabolite of AEA, the expression of other free fatty acids was also detected by LC-MS/MS. MUFAs with carbon chains from C14 to C24 and most PUFAs showed a high unitive increase, while among the SFAs, only C18:0 and C24:0 FFAs were increased in the CRC tissues. Increased MUFAs provide enough resource to build more phospholipids, diacylglycerols and triacylglycerols, to serve as a continuous nutrient fuel for rapidly proliferating cancer cells (
In conclusion, the present study demonstrated an increase in AEA in human CRC tissues along with lymph node metastasis, which could be an endogenous bio-indicator for the malignant degrees of CRC. Overexpression of its preferred receptor, CB1, aided in further understanding the important role of the endocannabinoid system in the treatment of human CRC. Moreover, the variation in levels of long chain ceramides and UFAs revealed that lipid dysfunction plays a vital role in CRC progression, and these levels may also be utilized as important monitoring biomarkers and novel drug targets for colorectal cancer.
The authors thank the Eye institute of xiamen University for assistance and Mrs Jin Fu for help with the design of this research. This study was supported by grants from the National Natural Sciences Foundation of China (no. 81373273), Fujian Provincial Natural Science Foundation (no. 2013J05122), and xiamen Southern Ocean Research Center Project (no. 14GYY018NF18).
Alteration of endocannabinoids and corresponding metabolic enzymes in human colorectal cancer tissues. (A) Level of AEA was increased in the CRC tissues. (B) Level of 2-AG showed no difference between the tumor tissues and the control. (C) The AEA metabolite, arachidonic acid, was upregulated in the CRC tissues. (D and E) The mRNA expression and enzyme activity of NPLD were enhanced in the CRC tissues. (F and G) The mRNA expression and enzyme activity of FAAH revealed the same upward trend in the CRC tissues. Non-tumor colon tissue controls (N, open bars), CRC with no lymph node metastasis (N0, gray bar) and CRC with lymph node metastasis (N1+, solid bars).
Varied levels of ceramides and ceramide synthases in the human colorectal cancer tissues. (A) C16:0, C24:0 and C24:1 ceramides were elevated, and C18:0/C20:0 ceramides were downregulated in the CRC tissues. (B) The mRNA levels of CerS1, CerS2, CerS5 and CerS6 were upregulated in the CRC tissues (N0, gray bar, CRC without lymph node metastasis; N1+, solid bars, CRC with lymph node metastasis) compared with the non-tumor colon tissue controls (N, open bars). *p<0.05, **p<0.01, ***p<0.001, n=22–47.
Levels of saturated fatty acids (SFAs) in human colon cancer tissues. No obvious variation of SFAs appeared, except for upregulated levels of C18:0 and C24:0 FFAs in CRC tissues without lymph node metastasis (N0, gray bar) and CRC with lymph node metastasis (N1+, solid bars), which were compared with the non-tumor colon tissue controls (N, open bars), *p<0.05, **p<0.01, n=22–47.
High expression levels of MUFAs in human colon cancer tissues. Levels of MUFAs with carbon chains from C14 to C24 were higher in the CRC tissues without lymph metastasis (N0, gray bar) and CRC tissues with lymph node metastasis (N1+, solid bars), which were compared with the non-tumor colon tissue controls (N, open bars), *p<0.05, **p<0.01, ***p<0.001, n=22–47. MUFAs, monounsaturated fatty acids.
Levels of PUFAs in human colon cancer tissues. Except for C18 PUFAs, levels of most PUFAs with carbon chains from C14 to C24 were higher in the CRC tissues without lymph metastasis (N0, gray bar) and CRC tissues with lymph metastasis (N1+, solid bars), which were compared with the non-tumor colon tissue controls (N, open bars). *p<0.05, **p<0.01, ***p<0.001, n=22–47. PUFAs, polyunsaturated fatty acids.
Expression levels of cannabinoid receptors in human colon cancer tissues. The protein expression levels (A) and mRNA expression levels (B) of CB1 and CB2 in the non-tumor colon tissue controls (N, open bars), CRC tissues without lymph metastasis (N0, gray bar) and CRC tissues with lymph node metastasis (N1+, solid bars), *p<0.05, ***p<0.001, n=22–47.
Characteristics of the CRC patients.
Characteristics | Data | |
---|---|---|
Age (years) | ||
Median | 60 | |
Range | 33–88 | |
Gender, n (%) | ||
Male | 31 | (66) |
Female | 16 | (34) |
Tumor stage, n (%) | ||
T2 | 2 | (4) |
T3 | 32 | (68) |
T4 | 13 | (28) |
Nodal status, n (%) | ||
N0 | 22 | (47) |
N1+ | 25 | (53) |
TNM staging, n (%) | ||
I | 1 | (2) |
II | 21 | (45) |
III | 20 | (42) |
IV | 5 | (11) |