Through the high-throughput measurement of endogenous metabolites, metabolomics has showed enormous prospects in discovering diagnostic cancer biomarkers in the field of renal cell carcinoma and bladder cancer. However, to the best of our knowledge, although many cancer markers have been found in bladder cancer, most researches only focused on the differentiation between cancers and healthy subjects, thus ignoring the discrimination within the malignant tumors. As we know, our study is the first one that explored the differential metabolites between BC and RCC, regardless of with or without hematuria. As a result, by comparing the BC, RCC and HCs, we found that: i) a panel made up of α-CEHC, β-cortolone, deoxyinosine, flunisolide, 11b,17a,21-trihydroxypreg-nenolone and glycerol tripropanoate could well distinguish the cancers (BC &RCC) from the healthy controls, which may provide significant information about the dysregulated metabolic pathways of malignant urinary tumors. ii) a panel consisiting of 4-ethoxymethylphenol, prostaglandin F2b, thromboxane B3, hydroxybutyrylcarnitine, 3-hydroxyphloretin and N'-formylkynurenine show a good ability to differentiate BC from RCC without hematuria. iii) Since previous studies have already indicated that hematuria may statistically affect the analytic outcomes of metabolomics, we also perform an exclusive experiment to certify the biomarker panel. As the result suggested, a panel comprising 1-hydroxy-2-oxopropyl tetrahydropterin, 1-acetoxy-2-hydroxy-16-heptadecyn-4-one, 1,2-dehydrosalsolinol and L-tyrosine could significantly discriminate BC from RCC among patients with hematuria.
The clustering heatmap between Cancers and Healthy controls suggested that the lipid metabolism was upregulated in Cancers, which was in accordance with the classical Warburg effect, demonstrating that cancer cells prefer to use glycolysis rather than aerobic oxidation even in the presence of oxygen22. As we know, the dysregulated lipid and phospholipid metabolism showed great significance in cel motality, cell invision and tumor metastasis, which may produce enormous tumor biomarkers23. In previous studies, the disturbance of lipid metabolism have been reported in various researches, including BC and RCC. By analysing the global lipidomic profiles of 165 bladder-derived tissues, Piyarathna etc. found that compared with benign tissues, the urothelial cancer of the bladder had higher levels of phospholipids and fatty acids, and reduced levels of triglycerides, which suggested that the reduced triglycerides may be used for producing energy, while the changed phospholipid may play an active role in membrane structure or signal transduction24. Bying performing comparative UPLC-MS of two isogenic human T24 bladder cancer cell lines, Young Lee et al. discovered that there was an statistically distinguished lipid species between cisplatin-sensitive and cisplatin-resistant cancer cells, suggesting that lipid-targeted new drug may improve the prognosis of cisplatin-resistant patients25. As for the RCC, an article reported that a lot of fatty acids were downregulated in nonmetastatic RCC tissue as a result of overly-active fatty acid oxidation. Besides, they also discovered that in metastatic RCC, the lipid metabolism was upregulated obviously, which may be related to the tumor progression26. In some other researches, metabolites of carnitine metabolism, which are responsible for the transportation of fatty acid into the mitochondria, have been found to be increased in high-grade tumor tissues, blood serum or urine27-29, which may be a consequence of improved fatty acid β-oxidation to sustain higher rates of cell division and growth.
2,5,7,8-tetramethyl-2(2'-carboxyethyl)-6-hydroxychroman(also known as α-CEHC), is an end-product of α-tocopherol, one group of vitamin E generated through a set of enzymatic reaction30. As we know, vitamin E is a potent lipid-soluable antioxident and it could help strengthen the immune systems, inhibit cell proliferation and several inflammation pathways caused by infection or tumor progression31,32. In our study, the α-CEHC is upregulated compared with the healthy controls and the fold change is 4.38, which confirms an acceralated vitamin E metabolism. To the best of knowledge, our study is the first one that discovered the upregulation of vitamin E metabolism in bladder caners, which may be caused by inflammation secondary to tumors. Concerning the renal cell carcinoma, Catchpole et al. observed that an increased level of α-tocopherol in RCC tumor tissues compared with the normal renal cortex tissue, consistent with the findings of Nikiforova etc 26,33. Besides, analysing the 66 invasive ovarian carcinomas and 9 borderline tumor tissues by gas chromatography/time-of-flight mass spectrometry, Denkert etc. discovered that α-tocopherol 2 was elevated in cancers and the fold change of cancer vs borderline tumor was 2.534. As all the vitamins in our bodies are gained through our digestation, we still can’t rule out the possibility that the increased vitamin metabolism may be just a superfical phenomenon of an increased uptake of lipids, rather than caused by the cancers.
A disturbance of the purine metabolism has also been detected in our study not only in the panel of cancer vs healthy controls but also in the group of BC vs RCC. However, contrary to most previous studies, our researches showed that compared with the controls, deoxyinosine, one of the most common precusor of DNA was decreased in cancer groups. In 2007, Sahu ec al. enrolled 96 samples(including 72 urothelial carcinoma patients and 24 normal patients) and analysed their differential metabolites by performing UHPLC- MS/MS. As a result, both the purine and the puring metabolites were increased in urothelial cancer, suggesting the accelerated synthesis and degradation of nucleotides35. In a meta-analysis of 11 articles, the levels of guanine, cytosine, thymine, hypoxanthine, uracil and ribose were found eleveted in the urine of BC patients, indicating a higher level of nucleotide metabolism36. Concerning the RCC, few researches reported the differential metabolites of purine metabolism, making our study the first one to demonstrate the inner machanism. Compred with the BC, the purine metabolism of RCC was upregulated slightly, which suggested a higher nuclein metabolism . However, it’s necessary to stess that the lowver purine level may be due to a much more obvious degradation together with a enhanced synthesis.
In our analysis, an perturbation of amino acid metabolism has also been revealed between the group of BC vs RCC with hematuria, namely alanine, aspartate, glutamate and D- glutamine metabolism, which suggested a distingushed protein metabolism between BC and RCC. Besides, the elevated prostaglandin F2b and thromboxane B3 occurred in BC, which are biologically active signaling components of the COX and LOX pathways. The COX and LOX pathways are closely associated with a function of inflammatory cell regulation, tumorigenesis, cell proliferation, and angiogenesis. Our results were supported by a previous metabolomic analysis of urothelial carcinoma35, illustrating a hyperactive tumor metabolism and the consequent inflammtion.
There also exist some limitations in our study. Firstly, the sample scale in our study is relatively small and it’s single-center, making it less convincible. Therefore, increasing the samples and enrolling more medical centers would be necessary in our further analysis. Secondly, our study focused on the discrimination of BC and RCC and revealed the deeper mechanism under the surface. However, due to the complete heterology of BC and RCC, it remains a question whether they are comparable. Beisdes, because of the epidemic difference between BC and RCC—the diagnostic age of BC is older than RCC, there is also a possiblitiy that the metabolic disturbance between BC and RCC may be caused by the distinguished age between the two groups, rather than by the cancer. Last but not least, owning to the limitations of time and conditions, we merely used one method — metabolomics to predict the potential altered metabolism and thus we only focused on the small metabolites in urine. Therefore, a combination of proteomics, transcriptomics and genomics in the future could help us better understand the deeper mechanism in BC and RCC.