Over the past decade, metabolomics studies have provided valuable information on the metabolic profile of patients suffering from various diseases, including cancer, and identified potential markers of developing or recurring disease. Cancer cells have the ability to reprogram their metabolism in order to support the increased need for energy caused by rapid proliferation. Monitoring of changes in the levels of various metabolites in cancer cells or body fluids may be a potential source of new cancer biomarkers. To date, many studies have been published indicating the high potential of metabolomic markers in the diagnosis of various cancers and in understanding of the mechanisms of cancer initiation and development 27.
In this study UHPLC-UHRMS and -UHRMS/MS methods were employed to evaluate changes in serum metabolite levels between 100 bladder cancer patients and 100 normal controls. The largest class of compounds differentiating the NCs group from the BC patients were lipids and lipid-like molecules. Lipids are the fundamental building blocks of all cell membranes and serve as a long-term energy storage. Furthermore, lipids have many other important functions within living organisms including transmit nerve impulses, production and regulation of certain hormones, cushion vital organs, intracellular signal transmission and cell transporting systems. Lipid metabolism is involved in various processes associated with cancer cells. Over the past decade, numerous studies have demonstrated that lipids and metabolites associated with lipid metabolism may be potential markers in human cancers including bladder cancer 28. We found that the plasma content of 10 glycerophospholipids including PE(P-16:0e/0:0), PC(16:1/16:1), PC(16:0/18:3), LPE(P-18:0/0:0), LPC(14:0/0:0), LPC(P-18:0), LPC(18:3), LPC(18:2), LPC(20:3), LPC(22:5) were significantly higher in the serum of NCs than in the BC subjects. This finding is in line with previous metabolomic studies that demonstrated an association of changes in the levels of these lipids in the blood with various cancers 29. Thus, alterations in these lipids’ metabolism may, therefore, play important roles in the development and progression of bladder cancer.
Glycerophospholipids (GPs), also called phospholipids include phosphatidylethanolamines (PE), phosphatidylcholines (PC) and phosphatidylethanolamines (PE), all of which are glycerol-based phospholipids. These compounds are a major component of the membranes of animal cells in which they are asymmetrically distributed acting as the matrix of different membrane proteins. Many previous studies have found low serum PE levels in various cancers including colon, prostate, lung, and breast cancers indicating these compounds as potential tumor markers 30,31. Serum levels of PE(P-16:0e/0:0) were found by Lin et. al significantly lower in patients with kidney cancer compared to controls 32. Some studies have provided evidence that translocation of PE from the inner to the outer leaflet of the plasma membrane indicating a loss of asymmetric distribution of aminophospholipids has been shown as the first sign of impending apoptosis. Thus, lower levels of PE(P-16:0e/0:0) in serum may be an early symptom of apoptotic cell death 33. Moreover, human phosphatidylethanolamine-binding protein is associated with resistance to apoptosis of tumor cells 34. It has been reported that exogenous PEs inhibits the growth and indicates an apoptosis of human hepatoma HepG2 cells 35. Lysophosphatidylethanolamine LPC, LysoPC), LysoPE (P-18:0/0:0) also known as LPE(18:0) was found in lower level in plasma of patients with liver, gastric colorectal, ovarian and lung cancer compared to the control group 31,36,37. Lysophosphatidylcholines (LPC, lysoPC) are an important endogenous signaling phospholipids involved in a variety of important processes, including cell migration, cell proliferation, inflammation and angiogenesis. Decreased LPC plasma level in cancer was also observed in previous study and was associated with body weight loss and increased inflammation. Level of these compounds is inversely correlated with C-reactive protein levels in plasma (CRP) 38. LPCs were found to be disturbed in several diseases including cancer. Previous metabolomic studies have reported lower level of PC(34:4), LysoPC(20:3) and LPC(P-18:0) in plasma of patients with ovarian cancer (EOC) compared to control 39. Zhang et al 40 have reported that LPC(14:0) was down-regulated in patients with recurrent EOC. Lower level of lysophospholipids have been associated with high activity of specific cell-surface G protein-coupled receptors which may cause apoptosis. Tan et al. 41 observed significantly lower of LPC(14:0) in the serum of patients with colorectal cancer compared with healthy controls. LPC(18:1), LPC(18:2) and LPC(18:3) were significantly decreasing in plasma of patients with colorectal cancer compared with healthy controls 42,43. Lee et al. 31 showed that the levels of LPC(18:2) were lower in plasma samples of patients with colorectal cancer and higher in plasma samples of patients with liver, gastric, lung and thyroid compared to those of healthy control individuals using UHPLC-MS/MS. LPC(18:2) was also found in lower level in plasma of patients with ovarian cancer compared to the control group 36. Previous metabolomic studies have demonstrated that LysoPC(18:1), LysoPC(20:3) were down-regulated in patients with ovarian cancer. Four of these compounds including LPC(14:0), LPC(18:3), LPC(20:3), LPC(22:5) were previously related to kidney injury. Metabolic profiling of plasma from patients with cancer cachexia revealed significantly lower levels of LPC(14:0), LPC(P-18:0), LPC(18:2), LPC(20:3), LPC(22:5) and LPE(18:0) compared to healthy controls 44. Three of these six LPC including LPC(18:1), LPC(18:3), LPC(22:5) we identified previously at lower levels in serum of patients with thyroid carcinoma 45. To our knowledge, only one lipid out of the ten most differentiating both groups cancer and control we indicated has been previously associated with bladder cancer. Tan et al. 46 indicated slightly higher level of LPC(18:2) in serum of patients with BC compared to controls using UHPLC-Q-ToF MS.
Lower levels of four prenol lipids including perillyl alcohol, D-limonene, thymol, alantolactone were found in serum of BC compared to controls. These monoterpenoids commonly occurring in many plants are known for their anti-tumor, antioxidant, anti-inflammatory and anti-fungal activity. Thymol and limonene have been shown to inhibit bladder cancer cell proliferation and induces these cells apoptosis 47,48.
We found that serum levels of metabolites: L-acetylcarnitine, linoleic acid and elaidic acid were higher and three others: valeric acid and 7-epi-jasmonic acid lower in BC patients compared to NCs. The levels of linoleic and elaidic acid were also found as significantly higher in patients with colorectal cancer 49. Increased serum activity of acetylcarnitine have been previously pointed out as a potential tumor biomarkers 50,51. Acetylcarnitine is a naturally occurring acetic acid ester of carnitine, important in mitochondrial tricarboxylic acid (TCA) cycle activity. Increased urine levels of this compound have previously been reported in patients with BC 52. Elevation of acetylcarnitine may be an indication of decreased carbon flow into the TCA cycle or excess production of acetyl-CoA 53. Previous studies revealed elevated urine level of acetylcarnitine and isovalerylcarnitine in BC patients compared to controls 54,55. However, the association between isovalerylcarnitine and bladder cancer has not yet been explained.
In order to apply the correct treatment regimens for BC patients, in addition to indicating the neoplasm, it is necessary to precisely and accurately indicate the stage and grade of this cancer. In total, 23 differential metabolites were identified as potential marker for discriminating between LG and HG BC patients and NCs. Among these metabolites, 18 metabolites were the common characteristic of both LG and HG BC patients. Three metabolites including lysoPC(20:3), PE(P-16:0e/0:0) and 2(4H)-benzofuranone, 6-Hydroxy-4,4,7a-trimethyl-5,6,7,7a-tetrahydrobenzofuran-2(4H)-one were identified in much higher level only in the serum of patients with HG BC, while four metabolites including 3-hexanone, diethylene glycol 2-ethylhexyl ether, elaidic acid, umbelliferone were found in significant higher level only in the serum of patients with LG BC patients.
In total, 38 differential metabolites were identified as potential marker for discriminating between pTa, pT1 and pT2 BC patients and NCs. Among these metabolites, 22 metabolites were the common to all three stages of BC. Two metabolites including Cys-Pro and curcumol were identified in much higher levels only in the serum of patients with pTa BC, while two metabolites including LysoPC(20:3) and alpha-hydroxyisobutyric acid were found in significant higher level only in the serum of patients with pT1 BC patients. Moreover, five metabolites including norcamphor, creatinine, dihydrojasmone, pro-leu, palmitoleoyl ethanolamide were found in significant higher level only in the serum of patients with pT2 BC patients.