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Metabolomic profiling identifies novel biomarkers and mechanisms in human bladder cancer treated with submucosal injection of gemcitabine

Metabolomic profiling identifies novel biomarkers and mechanisms in human bladder cancer treated... INTERNATIONAL JOURNAL OF MOLEc ULAR MEd Ic INE 44: 1952-1962, 2019 Metabolomic profiling identifies novel biomarkers and mechanisms in human bladder cancer treated with submucosal injection of gemcitabine 1* 2,3* 1* 2,3 2,3 cHAO YANG , XIAN SUN , HENGBING WANG , TING LU , KEQING WU , 2,3 1 4 5 6 YUSHENG GUAN , JING TANG , JIAN LIANG , RONGLI SUN , ZHONGYING GUO , 7 8 1 1 1 1 SINIAN ZHENG , XIAOLI WU , HESONG JIANG , XI JIANG , BING ZHONG , XIAOBING NIU , 6 2,3 2,3 1 SUAN SUN , XINRU WANG , MINJIAN cHEN and GUANGBO FU Department of Urology, The Affiliated Huai'an No. 1 People's Hospital of Nanjing Medical University, Huai'an, Jiangsu 223300; State Key Laboratory of Reproductive Medicine, Center for Global Health; Key Laboratory of Modern Toxicology of Ministry of Education, School of Public Health, Nanjing Medical University, Nanjing, Jiangsu 211166; Center of Reproduction and Genetic, The Affiliated Huai'an No. 1 People's Hospital of Nanjing Medical University, Huai'an, Jiangsu 223300; Key Laboratory of Environmental Medicine Engineering, Ministry of Education, School of Public Health, Southeast University, Nanjing, Jiangsu 210009; Department of Pathology, The Affiliated Huai'an No. 1 People's Hospital of Nanjing Medical University, Huai'an, Jiangsu 223300; department of Urology, Ningbo Medical Center Lihuili Eastern Hospital, Ningbo, Zhejiang 315040; Department of Pharmacy, The Affiliated Huai'an No. 1 People's Hospital of Nanjing Medical University, Huai'an, Jiangsu 223300, P.R. China Received April 18, 2019; Accepted September 6, 2019 d OI: 10.3892/ijmm.2019.4347 Abstract. Bladder cancer (Bc a) is a common urinary tract resection of bladder tumor (TURBT) may prevent recurrence malignancy with frequent recurrences after initial resection. of urothelial cancer. However, the underlying mechanism Submucosal injection of gemcitabine prior to transurethral remains unknown. In the present study, ultra-performance liquid chromatography Q-Exactive mass spectrometry was used to profile tissue metabolites from 12 BCa patients. The 48 samples included pre- and post-gemcitabine treatment Bc a tissues, as well as adjacent normal tissues. Principal Correspondence to: Professor Guangbo Fu, d epartment of component analysis (Pc A) revealed that the metabolic Urology, The Affiliated Huai'an No. 1 People's Hospital of Nanjing profiles of pre‑gemcitabine BCa tissues differed signic fi antly Medical University, 1 West Huanghe Road, Huai'an, Jiangsu 223300, from those of pre-gemcitabine normal tissues. A total of P.R. c hina E-mail: fgb200@vip.163.com 34 significantly altered metabolites were further analyzed. Pathway analysis using MetaboAnalyst identified three Professor Minjian c hen, State Key Laboratory of Reproductive metabolic pathways closely associated with Bc a, including Medicine, c enter for Global Health, School of Public Health, glutathione, purine and thiamine metabolism, while gluta- Nanjing Medical University, 818 East Tianyuan Road, Nanjing, thione metabolism was also identified by the enrichment Jiangsu 211166, P.R. c hina E-mail: minjianchen@njmu.edu.cn analysis using MetaboAnalyst. In search of the possible targets of gemcitabine, metabolite profiles were compared c ontributed equally between the pre-gemcitabine normal and post-gemcitabine Bc a tissues. Among the 34 metabolites associated with Abbreviations: TURBT, transurethral resection of bladder Bc a, the levels of bilirubin and retinal recovered in Bc a tumor; PCA, principal component analysis; ACN, acetonitrile; tissues treated with gemcitabine. When comparing normal KEGG, Kyoto Encyclopedia of Genes and Genomes; HMDB, bladder tissues with and without gemcitabine treatment, human metabolome database; SMPDB, Small Molecule Pathway among the 34 metabolites associated with Bc a, it was Database; HPLC/MS, high‑performance liquid chromatography/ observed that histamine change may be associated with mass spectrometry; UPLC‑MS, ultra‑performance LC‑MS; the prevention of relapse, whereas thiamine change may be UPLC‑TOF‑MS, UPLC time‑of‑flight MS; UPLC‑HRMS, UPLC involved in possible side effects. Therefore, by employing a high‑resolution MS; BCa, bladder cancer hypothesis-free tissue-based metabolomics study, the present Key words: tissue metabolomics, submucosal injection, gemcitabine, study investigated the metabolic signatures of Bc a and found biomarkers, bladder cancer that bilirubin and retinal may be involved in the mechanism underlying the biomolecular action of submucosal injection of gemcitabine in urothelial Bc a. YANG et al: METABOLOMIc ANALYSIS OF GEM c ITABINE-TREATEd BLA dd ER c ANc ER Introduction Huai'an No. 1 People's Hospital of Nanjing Medical University were recruited between d ecember 2016 and September 2017. Bladder cancer (Bc a) ranks ninth among the most common Bladder tissue samples were collected from the same patient solid tumors worldwide (1). Approximately 75% of newly immediately prior to and 30 min after submucosal injection diagnosed Bc a cases are non-muscle invasive, and the majority of gemcitabine (50 mg, dissolved in 20 ml normal saline). The are histologically low-grade cancer (2). Routine surveillance bladder tissues included Bc a as well as adjacent non-cancerous to monitor Bc a recurrence includes cystoscopic examination bladder tissues. Therefore, a total of 48 samples were collected due to a high risk of recurrence after initial resection. The repeat in four groups: Pre-gemcitabine normal, pre-gemcitabine transurethral resection of bladder tumor (TURBT) remains the Bc a, post-gemcitabine normal, and post-gemcitabine Bc a. first‑line treatment for BCa recurrence (3). However, all these Histopathological diagnosis was conducted by two indepen- invasive procedures result in a high cost of care, and are often dent pathologists according to the classic fi ation criteria of the associated with signic fi ant morbidity. Therefore, more effective World Health Organization/International Society of Urological interventions to prevent Bc a recurrence are urgently needed. Pathology (21). Written informed consent was obtained from Submucosal injection of antitumor drugs (pirarubicin) each participant prior to recruitment. The Ethics c ommittee after standard TURBT was proven to be an effective approach of The Affiliated Huai'an No. 1 People's Hospital of Nanjing to reducing superc fi ial tumor recurrence (4). Gemcitabine is Medical University reviewed and approved the study protocol a pivotal chemotherapeutic agent widely used for Bc a due to (serial no. YL‑P‑2013‑21‑01). The sample size of the present its low toxicity in general (5) and as an intravesical instilla- study was similar to that of a previous study on Pc a tissues, tion (3). d ata from our experimental and clinical studies also which produced valuable findings (22). In addition, the demonstrated that submucosal injection of gemcitabine prior experimental design of self-control and complete collection to TURBT signic fi antly reduced BCa recurrence (6). However, of samples in our study may improve statistical power by the underlying mechanisms are largely unknown. avoiding confounding factors. Metabolomics is a newly emerging technology, which enables the identification of endogenous compounds and Tissue preparation for metabolomic analysis. Following potentially novel mechanisms associated with disease harvesting, all tissues were snap‑frozen in liquid nitrogen, and processes (7). Metabolomics has been used to profile kept in a ‑80˚C freezer until further analysis. The sample prep - metabolites in various biological samples, such as serum (8), aration was conducted as described previously (23). Briey fl , urine (9-15) and tissue (16,17), which are the results of the the tissues were fragmented, ultrasonicated for 5 min (power: metabolic response of living systems to drug toxicity or 60%, pulses: 6/4) in distilled water, and then 150 µl homog- disease (10). Potential biomarkers identified from metabo - enate and 450 µl methanol (Merck KGaA) were mixed in a lomic profiling studies on BCa may be of diagnostic value 1.5-ml Eppendorf tube for protein precipitation. The mixture and act as indicators of cancer recurrence (18). c urrently, a was centrifuged at 16,000 x g for 15 min at 4˚C, and the number of analytical platforms, such as high-performance supernatant was collected and dried in a vacuum centrifugal liquid chromatography/mass spectrometry (HPLc /MS) (10), concentrator. The dry residue was reconstituted in ultra-pure ultra-performance Lc- MS (UPLc- MS) (12), and UPLc water and used for metabolomic analysis. time‑of‑flight MS (UPLC‑TOF‑MS) (15), have been employed to study the metabolomics of Bc a by using urine samples. Metabolomic analysis. The metabolomic analysis was However, metabolomic studies on Bc a tissues is relatively conducted as previously reported (23). Lc- HRMS analysis scarce (16,17). Notably, different metabolomic platforms with was performed on an UPLc Ultimate 3000 system (d ionex), their unique analytical approaches provide complementary coupled with a Q-Exactive mass spectrometer (Thermo Fisher insights into metabolome changes (9,11-15). Therefore, there is Scientic fi , Inc.). The instrument operated at a 70,000 resolution a need to study the tissue-based metabolic signatures of Bc a with a full-scan acquisition ranging from 70 to 1,500 m/z. The using a new metabolomics platform. chromatographic separation of metabolites associated with the Metabolomics has also been applied to cancer treatment metabolomic profiling used a multistep gradient containing and drug target discovery. Eidelman et al reported using ultra‑pure water (mobile phase A) and acetonitrile (ACN; metabolomics to screen the potential therapeutic pathways in mobile phase B), both acidified with 0.1% formic acid. The prostate cancer (Pc a) (19). Metabolomics has also been proven gradient operated at a o fl w rate of 0.4 ml/min over a 15-min to be a promising approach to developing reliable therapeutic period. The metabolites were identie fi d based on the accurate targets for Pc a treatment (20). The present study employed mass and the retention time compared with the commercial liquid chromatography (Lc) -Q-Exactive MS-based metabo- standards. The metabolite standards were purchased from lomic technology to study the metabolic changes in Bc a tissues Sigma‑Aldrich; Merck KGaA, Damas‑beta Co., Ltd., Aladdin before and after treatment with gemcitabine. Identic fi ation of Reagent company and Adamas Reagent co., Ltd. the key metabolites may reveal new metabolic changes associ- ated with Bc a and uncover the changes that mediate the effect Statistical analysis. d ata collected from the mass spec- of gemcitabine in the treatment of Bc a. trometer were processed for pattern recognition analysis (principal component analysis, PCA). Normalized MS data Materials and methods were exported to SIMc A-P+ software (V14.0, Umetrics AB) to perform Pc A where grouping trends could be observed. The Clinical samples. A total of 12 patients (9 men and 3 women; age difference in metabolites between two groups was compared range, 55‑85 years) who had undergone TURBT at the Affiliated by paired t-test. According to previous reports (24,25), the INTERNATIONAL JOURNAL OF MOLEc ULAR MEd Ic INE 44: 1952-1962, 2019 correlation between metabolite changes and cancer stage was (Table IV and Fig. 2B). The metabolic network of the differen- analyzed based on the comparison between two groups using tial metabolites and altered metabolic pathways in the KEGG paired t‑test based on the cancer stage classic fi ation. A P‑value general metabolic pathway map is shown in Fig. 2c . of <0.05 was considered as the threshold for statistically significant differences. Candidate targets of submucosal injection of gemcitabine. To further characterize the metabolic changes and the Based on targeted metabolomic analysis, we next analyzed these metabolic pathways involved, the differentiated metabolites 34 differential metabolite changes in post-gemcitabine Bc a were first annotated with Kyoto Encyclopedia of Genes and tissues, and compared their levels with those in pre-gemcitabine Genomes (KEGG, http://www.genome.jp/kegg/) and Human normal tissues. A total of 32 metabolites maintained signifi - Metabolome d atabase (HMd B, http://www.hmdb.ca/) (date cant changes with identical trends in the comparison between of access for databases: April 19, 2018). d ata were then pre-gemcitabine normal vs. pre-gemcitabine Bc a tissues, processed and analyzed using MetaboAnalyst 4.0 (http://www. indicating that the findings for differential metabolites in metaboanalyst.ca/MetaboAnalyst/) by R software (v3.4.3, Bc a are reliable. Importantly, the significant decrease in GitHub). Two modules of MetaboAnalyst were used, namely the levels of two metabolites associated with Bc a recovered pathway analysis and enrichment analysis, which are based on to insignificant levels following submucosal injection of the KEGG database and Small Molecule Pathway d atabase gemcitabine (Table II and Fig. 3A, B and E-F). These were (SMPd B, http://smpdb.ca/), respectively (26). The metabolic bilirubin and retinal, which may be the candidate targets of network of the differential metabolites and altered metabolic gemcitabine for the prevention of recurrence of urothelial pathways in KEGG general metabolic pathway was visualized BCa. We further analyzed the changes in the two metabolites by iPath 3.0 (http://pathways.embl.de/). in association with cancer stage. In Ta/T1 stage disease, when comparing pre-gemcitabine normal vs. pre-gemcitabine Bc a Results tissues, bilirubin was decreased significantly in the Bc a tissues (P=4.15E-2) (Table SI and Fig. 3c), while this decrease Clinical characteristics of 12 subjects. The mean age of the recovered to an insignificant level following submucosal subjects, including 9 men and 3 women, was 67 years (range, injection of gemcitabine (P=5.40E‑1) (Table SI and Fig. 3D); 55-85 years). In all 12 patients, the diagnosis of urothelial however, at this stage, the decrease of retinal in the tumor was carcinoma was confirmed by histopathological examination. still not statistically signic fi ant (P= 4.76E‑1) (Table SI). In T2 A total of 2 patients had T , 1 patient had T , and 9 patients had stage disease, when comparing pre-gemcitabine normal vs. a 1 T disease. Two patients were diagnosed with high-grade T pre‑gemcitabine BCa tissues, retinal was decreased signifi - 2 2 tumors with squamous metaplasia. The clinical characteristics cantly in the Bc a tissues (P=4.98E-2) (Table SI and Fig. 3G), of the participants are shown in Table I, and are considered to while its change became statistically insignic fi ant following be representative according to the general population of Bc a submucosal injection of gemcitabine (P=3.22E-1) (Table SI in c hina (2,27). and Fig. 3H); however, at this stage, the decrease of retinal in tumor was not statistically signic fi ant (P=1.68E‑1) (Table SI). PCA. A total of 165 metabolites were detected. Pc A was These results indicate that bilirubin and retinal changes were performed to process the metabolite data based on a mean correlated with cancer stage, and gemcitabine may exert its center-scaling model, which is an unsupervised projection effect through metabolic pathways associated with cancer method employed to visually display the intrinsic similari- stage. ties and differences in the dataset. As shown in Fig. 1, Pc A (pre-gemcitabine normal vs. pre-gemcitabine Bc a tissues) Effects of submucosal injection of gemcitabine on normal revealed a well-differentiated and clustered pattern in score tissues. To identify the effects of submucosal injection of plots, indicating the signic fi ant metabolome changes between gemcitabine on normal bladder tissues, the metabolomes of these two groups. normal tissues were compared pre- and post-gemcitabine treat- ment. A total of 10 metabolites were found to be signic fi antly Altered metabolites and cancer‑associated metabolic altered in the normal tissues following submucosal injection of pathways in BCa. UPLC‑Q‑Exactive analysis identified 34 gemcitabine, whereas only 2 metabolites were associated with differentially expressed metabolites annotated in the KEGG Bc a (Table V). Histamine was signic fi antly decreased in BCa, and HMd B databases in pre-gemcitabine Bc a tissues and its level was signic fi antly increased in normal tissues after compared with pre-gemcitabine adjacent normal tissues submucosal injection of gemcitabine (Fig. 3I and J). However, (Table II). thiamine was signic fi antly decreased in BCa, and its level was The 34 Bc a-associated metabolites were then submitted significantly decreased in normal tissues after submucosal to MetaboAnalyst for analysis. Table II lists all metabolites injection of gemcitabine (Fig. 3M and N). We further analyzed found to be altered in Bc a. Table III and Fig. 2A show the the changes in the two metabolite in relation to cancer stage. In metabolic pathways connected with these 34 metabolites, Ta/T1 stage disease, when comparing pre-gemcitabine normal among which three pathways, namely glutathione, purine vs. pre-gemcitabine Bc a tissues, the changes in histamine and thiamine metabolism, were signic fi antly associated with (P=2.60E-1) and thiamine (P=8.39E-2) in the tumor were Bc a. Furthermore, in order to expand our understanding of insignic fi ant (Table SI). However, in T2 stage disease, when metabolic pathways related to Bc a, the module of enrich- comparing pre-gemcitabine normal vs. pre-gemcitabine Bc a ment analysis of MetaboAnalyst was used, which verie fi d that tissues, the decrease in histamine (P=4.61E-3) (Table SI and glutathione metabolism was signic fi antly associated with BCa Fig. 3K) and thiamine (P=4.13E-4) (Table SI and Fig. 3O) in YANG et al: METABOLOMIc ANALYSIS OF GEM c ITABINE-TREATEd BLA dd ER c ANc ER Table I. clinical characteristics of 12 Bca patients. Histopathology --------------------------------------------------------------------------- Number Sex Age (years) Tumor type Stage Grade 01 Male 55 MIUc T2 High 02 Male 56 MIUc T2 High 03 Male 59 MIUc T2 High 04 Female 82 MIUc T1 High 05 Male 67 MIUc T2 High 06 Male 76 MIUc T2b Squamous metaplasia 07 Male 76 MIUc T2 High 08 Female 60 MIUc T2 High 09 Male 67 NMIUc Ta High 10 Male 58 NMIUc Ta High 11 Male 85 MIUc T2a High 12 Female 61 MIUc T2 Squamous metaplasia MIUC, muscle‑invasive urothelial carcinoma; NMIUC, non‑muscle‑invasive urothelial carcinoma. BCa, bladder cancer. Figure 1. Pc A score plots derived from pre-gemcitabine normal (green) and pre-gemcitabine Bc a (blue) tissues. c omparison of pre-gemcitabine normal (green) and pre‑gemcitabine BCa (blue) tissues. PCA, principal component analysis; BCa, bladder cancer. the BCa tissues was signic fi ant. In T2 stage disease, the hista - (P=4.11E-2) (Table SI and Fig. 3P). These results indicate that mine level was signic fi antly increased in normal tissues after these metabolite changes were correlated with cancer stage, submucosal injection of gemcitabine (P=3.55E-3) (Table SI and gemcitabine may exert its effects mainly through these and Fig. 3L), while thiamine was significantly decreased metabolic pathways in T2 stage disease. INTERNATIONAL JOURNAL OF MOLEc ULAR MEd Ic INE 44: 1952-1962, 2019 Table II. List of the altered metabolites identified in BCa and their changes in the comparison between BCa tissues with or without gemcitabine pretreatment and pre-gemcitabine normal tissues. Post-gemcitabine Bca Pre-gemcitabine Bca vs. pre-gemcitabine a a vs. normal tissues normal tissues -------------------------------------------------- ------------------------------------------------- a a Metabolites KEGG HMdB Fold change P-value Fold change P-value deoxycytidine c00881 HMdB0000014 4.03 1.22E-02 10.10 4.15E-05 5'‑Methylthioadenosine C00170 HMDB0001173 15.16 2.38E‑04 12.22 4.16E‑04 3'‑AMP C01367 HMDB0003540 4.95 3.02E‑02 6.36 3.33E‑02 Androstenedione c00280 HMdB0000053 0.16 4.09E-02 0.10 5.76E-03 Bilirubin C00486 HMDB0000054 0.4 3.97E‑02 1.26 2.04E‑01 cholic acid c00695 HMdB0000619 0.29 3.14E-02 0.24 2.00E-03 cytidine c00475 HMdB0000089 3.83 2.43E-03 3.05 6.77E-03 5-Hydroxylysine c16741 HMdB0000450 0.64 4.40E-02 0.48 7.49E-03 deoxyinosine c05512 HMdB0000071 2.44 3.87E-02 4.12 1.70E-02 Glucosamine 6-phosphate c00352 HMdB0001254 6.04 6.26E-03 6.87 3.02E-05 Glyceraldehyde c02154 HMdB0001051 0.29 5.86E-04 0.32 1.65E-03 Sphingosine c00319 HMdB0000252 2.03 3.30E-02 3.34 2.18E-05 Glycerophosphocholine c00670 HMdB0000086 56.88 1.93E-03 51.03 3.44E-03 Glycine c00037 HMdB0000123 0.55 2.22E-02 0.47 4.68E-03 Guanidine c17349 HMdB0001842 0.47 5.25E-03 0.01 3.79E-04 Hexadecanedioic acid c19615 HMdB0000672 0.27 1.34E-02 0.23 3.44E-03 Histamine c00388 HMdB0000870 0.58 2.49E-02 0.35 7.49E-03 Hypotaurine c00519 HMdB0000965 0.07 4.19E-04 0.08 5.33E-04 Inosinic acid c00130 HMdB0000175 92.72 1.73E-05 150.57 1.43E-03 L-carnitine c00318 HMdB0000062 2.24 2.39E-03 2.70 3.97E-03 L-cystine c00491 HMdB0000192 0.49 2.70E-02 0.13 1.17E-03 L-Phenylalanine c00079 HMdB0000159 0.51 2.80E-02 0.31 4.70E-03 N-Acetylneuraminic acid c19910 HMdB0000230 2.68 2.13E-02 2.90 3.38E-02 Oxidized glutathione C00127 HMDB0003337 13.97 4.36E‑03 16.05 2.77E‑03 L-Palmitoylcarnitine c02990 HMdB0000222 3.59 1.88E-02 5.09 2.77E-04 Pantothenol c05944 HMdB0004231 0.44 3.12E-02 0.18 8.44E-05 Pyroglutamic acid c01879 HMdB0000267 0.29 1.85E-03 0.16 5.30E-04 Quinic acid c06746 HMdB0003072 0.09 4.05E-02 0.25 4.76E-02 Retinal C00376 HMDB0001358 0.13 4.98E‑02 0.43 3.81E‑01 Rhamnose c00507 HMdB0000849 0.22 2.90E-03 0.17 2.40E-04 deoxycholic acid glycine conjugate c05464 HMdB0000631 0.19 6.50E-03 0.15 4.52E-04 Sorbitol c00794 HMdB0000247 0.29 9.19E-03 0.13 1.44E-03 Tetradecanedioic acid c11002 HMdB0000872 0.44 4.46E-02 0.36 4.47E-02 Thiamine c00378 HMdB0000235 0.34 6.95E-05 0.18 2.47E-05 In the fold change calculation, the metabolite in pre-gemcitabine normal tissues served as the denominator. Bold print indicates statistical significance. BCa, bladder cancer; KEGG, Kyoto Encyclopedia of Genes and Genomes; HMDB, human metabolome database. Discussion glutathione metabolism was the consistently altered pathway in enrichment and pathway analyses (Tables III and IV, and In the present study, UPLc- Q-Exactive-based metabolomic Fig. 2A and B); iii) among the 34 cancer‑associated metabolites, analysis was utilized to profile metabolites in BCa tissues. the levels of bilirubin and retinal recovered after gemcitabine The major findings may be summarized as follows: i) A total injection, suggesting that these two are likely the targets of of 34 key metabolites associated with BCa were identified gemcitabine treatment (Table II, Fig. 3A‑D and E‑H); iv) the (Table II); ii) three metabolic pathways, namely glutathione, effects of gemcitabine on normal bladder tissues were also purine and thiamine metabolism, were altered in Bc a, and investigated, and it was deduced that histamine may have the YANG et al: METABOLOMIc ANALYSIS OF GEM c ITABINE-TREATEd BLA dd ER c ANc ER Table III. Pathway analysis of metabolite changes in Bca . KEGG pathway Total Hits P-value Glutathione metabolism 38 3 1.55E-02 Purine metabolism 92 4 3.86E-02 Thiamine metabolism 24 2 4.40E-02 Nitrogen metabolism 39 2 1.04E-01 Primary bile acid biosynthesis 47 2 1.41E-01 Fructose and mannose metabolism 48 2 1.46E-01 cysteine and methionine metabolism 56 2 1.86E-01 cyanoamino acid metabolism 16 1 2.04E-01 Pyrimidine metabolism 60 2 2.07E-01 Taurine and hypotaurine metabolism 20 1 2.48E-01 Retinol metabolism 22 1 2.70E-01 Ether lipid metabolism 23 1 2.80E-01 Aminoacyl-tRNA biosynthesis 75 2 2.86E-01 Alanine, aspartate and glutamate metabolism 24 1 2.90E-01 Sphingolipid metabolism 25 1 3.01E-01 Pantothenate and coA biosynthesis 27 1 3.20E-01 Phenylalanine, tyrosine and tryptophan biosynthesis 27 1 3.20E-01 Methane metabolism 34 1 3.86E-01 Glycerophospholipid metabolism 39 1 4.28E-01 Porphyrin and chlorophyll metabolism 104 2 4.36E-01 Galactose metabolism 41 1 4.45E-01 Histidine metabolism 44 1 4.68E-01 Phenylalanine metabolism 45 1 4.76E-01 Lysine degradation 47 1 4.91E-01 Glycine, serine and threonine metabolism 48 1 4.98E-01 Fatty acid metabolism 50 1 5.13E-01 Amino sugar and nucleotide sugar metabolism 88 1 7.21E-01 Steroid hormone biosynthesis 99 1 7.63E-01 The analysis was conducted by the module of pathway analysis of MetaboAnalyst 4.0. BCa, bladder cancer; KEGG, Kyoto Encyclopedia of Genes and Genomes. ability to protect against disease recurrence, whereas thiamine altered oxidized glutathione in BCa; they discovered four may be involved in the side effects of treatment (Table V, single-nucleotide polymorphisms in the glutathione synthetase Fig. 3I‑L and M‑P), which requires further confirmation in gene, and these changes were associated with Bc a recurrence future studies. after TUR and Bacillus c almette Guerin treatment (31). Our The identic fi ation of the three metabolic pathways signifi - findings, together with others, suggest that oxidative stress in cantly altered in Bc a may be pathophysiologically important. Bc a cells is at least partially due to the disrupted glutathione Glycine is an important amino acid that participates in all metabolism. three metabolic pathways. A decrease in glycine was reported By using Lc- Q-Exactive MS, this study is, to the best of as a biomarker in Bc a (14). As a precursor of purine synthesis, our knowledge, the first to investigate the metabolite changes reduced glycine in Bc a indicates that a critical metabolic in Bc a treated with gemcitabine followed by TURBT. Among process associated with cell proliferation is altered in Bc a (14). the 34 cancer-associated metabolites, the levels of bilirubin and Notably, glutathione metabolism is the consistently altered retinal recovered after gemcitabine treatment, indicating that pathway in enrichment and pathway analysis. Glutathione they may be the potential targets of gemcitabine for reducing is the most abundant low-molecular-weight peptide present Bc a recurrence. Bilirubin, a degradation product of free heme in eukaryotic cells (28). Glutathione is a primary cellular groups, protected LLc- PK1 cells against cisplatin-induced antioxidant that effectively scavenges free radicals and other death (32). A large population-based study demonstrated reactive oxygen species (29) and, therefore, plays an important that patients with primary biliary cirrhosis (PBc) have a role in protecting cells from oxidative injury (30). Glutathione nine-fold increased risk of developing urinary Bc a, and Bc a is also involved in cellular detoxic fi ation, and is required in and PBc share a number of etiological factors (33). Uridine several aspects of the immune response (31). Ke et al reported 5'‑diphospho‑glucuronosyltransferases (UGTs) are enzymes INTERNATIONAL JOURNAL OF MOLEc ULAR MEd Ic INE 44: 1952-1962, 2019 Figure 2. Results of the metabolic connection analysis of the changed metabolomic data in Bc a. (A) Pathway analysis based on the KEGG database. (B) Enrichment analysis based on SMPd B. (c ) Metabolic network of the differential metabolites and altered metabolic pathways in KEGG general metabolic pathway map. Red dots represent the increased metabolites in BCa; green dots represent the specic fi ally decreased metabolites in BCa; orange line, glutathione metabolism; purple line, purine metabolism; blue line, thiamine metabolism. The metabolites and pathways not indicated in the general pathway map are not shown. The original general metabolic pathway map is available at https://pathways.embl.de/ipath3.cgi. KEGG, Kyoto Encyclopedia of Genes and Genomes; SMPDB, Small Molecule Pathway Database; BCa, bladder cancer. that participate in several biological processes involving Bc a (37). A high dietary intake of vitamin A reduces the bilirubin conjugation. UGTs catabolize carcinogens and, there- incidence of BCa (38). Ziouzenkova et al reported retinal as fore, protect bladder cells from the harmful effects of toxic a distinct biological regulator involved in suppressing adipo- chemicals accumulated in the bladder. Targeting UGT1A may genesis, diet-induced obesity and insulin resistance (39). The serve as a novel therapeutic intervention against uroepithelial potential effect of bilirubin and retinal on the clinical outcome carcinomas (34). Retinal (retinaldehyde; Rald) is derived from of patients with BCa identie fi d in the present study is worthy retinol (vitamin A) oxidized by alcohol dehydrogenases (35). of further investigation in the future. Retinal plays an essential role in molecular signaling in The effects of gemcitabine on the metabolism of adjacent vision, and serves mainly as a retinoic acid (an active form of normal tissues were also examined. Most gemcitabine-induced vitamin A) precursor outside the eye (36). The serum levels metabolites do not overlap with those identie fi d in BCa, indi - of vitamin A were found to be decreased in patients with cating that gemcitabine did not exert notable adverse effects on YANG et al: METABOLOMIc ANALYSIS OF GEM c ITABINE-TREATEd BLA dd ER c ANc ER Table IV. Enrichment analysis of metabolite changes in Bca . Pathway from SMPdB Total Hits P-value Glutathione metabolism 21 3 2.96E-02 carnitine synthesis 22 2 1.64E-01 Bile acid biosynthesis 65 4 1.64E-01 Glutamate metabolism 49 3 2.19E-01 Thiamine metabolism 9 1 2.63E-01 Fructose and mannose degradation 32 2 2.88E-01 Amino Sugar metabolism 33 2 3.00E-01 Pyrimidine metabolism 59 3 3.11E-01 Taurine and hypotaurine metabolism 12 1 3.35E-01 Retinol metabolism 37 2 3.50E-01 Porphyrin metabolism 40 2 3.87E-01 Fatty acid metabolism 43 2 4.23E-01 Methionine metabolism 43 2 4.23E-01 Alanine metabolism 17 1 4.39E-01 Beta oxidation of very long-chain fatty acids 17 1 4.39E-01 Purine metabolism 74 3 4.51E-01 Spermidine and spermine biosynthesis 18 1 4.58E-01 Pantothenate and coA biosynthesis 21 1 5.11E-01 Androstenedione metabolism 24 1 5.59E-01 Glycerolipid metabolism 25 1 5.74E-01 Oxidation of branched chain fatty acids 26 1 5.89E-01 Mitochondrial beta-oxidation of short-chain saturated fatty acids 27 1 6.03E-01 Mitochondrial beta-oxidation of long-chain saturated fatty acids 28 1 6.16E-01 Phenylalanine and tyrosine metabolism 28 1 6.16E-01 Ammonia recycling 32 1 6.66E-01 Androgen and estrogen metabolism 33 1 6.78E-01 Aspartate metabolism 35 1 6.99E-01 Galactose metabolism 38 1 7.29E-01 Sphingolipid metabolism 40 1 7.48E-01 Histidine metabolism 43 1 7.73E-01 Arginine and proline metabolism 53 1 8.41E-01 Glycine and serine metabolism 59 1 8.72E-01 Tryptophan metabolism 60 1 8.76E-01 Arachidonic acid metabolism 69 1 9.10E-01 The analysis was conducted by the module of enrichment analysis of MetaboAnalyst 4.0. Bca, bladder cancer. SMPdB, Small Molecule Pathway database. normal bladder tissues. It was observed that histamine change was signic fi antly higher compared with that in rats fed bracken may be associated with the prevention of relapse. Histamine fern but receiving no thiamine supplements, as thiamine may is derived from the decarboxylation of histidine by histidine interfere with the absorption, distribution, metabolism, or decarboxylase in mammals (8). Histamine is primarily released excretion of the bracken fern (43). However, a case-control in ina fl mmatory processes by mast cells (8), which are closely study from New Hampshire investigated the effect of minerals associated with Bc a (40). Histamine H1 receptor (HRH1) and vitamins on the risk of Bc a, and found that a higher total expression was identie fi d in BCa and found to be associated intake of thiamine was inversely correlated with Bc a risk in with the prognosis (41). It was observed that thiamine change older participants (44). may be involved in treatment-related side effects. Thiamine, There is currently a lack of effective biomarkers for Bc a or vitamin B-1, is a water-soluble vitamin (42). An early report diagnosis and prognosis. Metabolomic profiles from tissue by Pamukcu et al demonstrated that the incidence of urinary have the potential to be used, along with other current diag- bladder carcinomas in rats fed bracken fern and additionally nostics, to help guide the clinical management of patients s.c. injected once weekly with 2 mg of thiamine hydrochloride with BCa. The changed metabolites identie fi d in the present INTERNATIONAL JOURNAL OF MOLEc ULAR MEd Ic INE 44: 1952-1962, 2019 Table V. changes in normal tissues after gemcitabine treatment. Pre-gemcitabine Post-gemcitabine vs. a a Bca vs. normal tissues pre-gemcitabine normal tissues ------------------------------------------------------------------- ------------------------------------------------------------------- a a Metabolites Fold-change P-value Fold-change P-value 3-Methyladenine 1.54 3.78E-01 0.17 2.33E-02 Ascorbic acid 22.26 1.85E-01 0.28 5.46E-03 creatinine 0.48 3.44E-01 0.13 1.16E-02 d-Glyceraldehyde 3-phosphate 0.72 3.33E-01 0.15 2.16E-02 Histamine 0.58 2.49E-02 2.26 3.73E-04 N-Acetylglutamic acid 0.52 8.58E-02 0.57 4.95E-02 N-Acetylglutamine 0.68 5.45E-01 0.11 4.25E-02 Thiamine 0.34 6.95E-05 0.65 2.11E-02 Tryptamine 0.10 7.66E-02 0.11 3.23E-02 Uridine 1.41 6.74E-02 1.51 3.65E-02 In the fold change calculation, the metabolite in pre-gemcitabine normal tissues served as the denominator. Bold print indicates statistically significant differences. BCa, bladder cancer. Figure 3. Relative levels of potential candidate targets of submucosal injection of gemcitabine (A, B, E, F, I, J, M and N). All samples; (C and D) Ta/T1 stage; (G, H, K, L, O and P) T2 stage. Values are presented as mean ± standard error of the mean. The P-values of paired t-test are indicated. YANG et al: METABOLOMIc ANALYSIS OF GEM c ITABINE-TREATEd BLA dd ER c ANc ER study, such as glycine, may be used as potential biomarkers Patient consent for publication for Bc a. In addition, those metabolites discovered in Bc a with gemcitabine treatment may reveal new metabolic pathways Not applicable. that mediate the anti-recurrence effect of gemcitabine, which currently remain elusive. Competing interests In summary, the present study employed UPLc- HRMS- based metabolomic analysis to investigate metabolite changes The authors declare that they have no competing interests. in bladder tissues from Bc a and Bc a treated with gemcitabine. The findings may provide new insights into metabolic changes Authors' information in Bc a and the biomolecular basis of submucosal injection of gemcitabine for Bc a. In addition, the study demonstrated Chao Yang: 15949177271@163.com; Xian Sun: 1019949991@ that the UPLc- HRMS-based metabolomic analysis provides qq.com; Hengbing Wang: wanghengbing2004@163.com; comprehensive metabolite profiling data that may pave the Ting Lu: 552095014@qq.com; Keqing Wu: wkwly1993@ way to a novel approach to Bc a research. qq.com; Yusheng Guan: guanys@njmu.edu.cn; Jing Tang: tjing19681222@sina.com; Jian Liang: lj3936@126.com; Acknowledgements Rongl i Su n: 101012172@seu. e du.cn; Z hong yi ng Guo: guozhongying407@163.com; Sinian Zheng: zhengsinian@163. Not applicable. com; Xiaoli Wu: wuxiaoli2233@126.com; Hesong Jiang: njjhs2007@163.com; Xi Jiang: piscesjxi@163.com; Bing Funding Zhong: 15152569186@163.com; Xiaobing Niu: bingke2008@ si na.com; Sua n Sun: hay yssa@163.com; X i n r u Wa ng: The present study was supported by grants from the National xrwang@njmu.edu.cn Natural Science Foundation (81872650 and 81573182); the Natural Science Foundation of the Jiangsu Higher Education References Institutions of China (18KJA320003 and 18KJB320001); 1. Ferlay J, Soerjomataram I, d ikshit R, Eser S, Mathers c , the Key Research & d evelopment Plan of Jiangsu Province Rebelo M, Parkin d M, Forman d and Bray F : c ancer incidence (BE2017628); the Southeast University & Nanjing Medical and mortality worldwide: Sources, methods and major patterns University Collaborative Research Project (2242018K3DN25); in GLOBOc AN 2012. Int J c ancer 136: E359-E386, 2015. 2. 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Metabolomic profiling identifies novel biomarkers and mechanisms in human bladder cancer treated with submucosal injection of gemcitabine

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Abstract

INTERNATIONAL JOURNAL OF MOLEc ULAR MEd Ic INE 44: 1952-1962, 2019 Metabolomic profiling identifies novel biomarkers and mechanisms in human bladder cancer treated with submucosal injection of gemcitabine 1* 2,3* 1* 2,3 2,3 cHAO YANG , XIAN SUN , HENGBING WANG , TING LU , KEQING WU , 2,3 1 4 5 6 YUSHENG GUAN , JING TANG , JIAN LIANG , RONGLI SUN , ZHONGYING GUO , 7 8 1 1 1 1 SINIAN ZHENG , XIAOLI WU , HESONG JIANG , XI JIANG , BING ZHONG , XIAOBING NIU , 6 2,3 2,3 1 SUAN SUN , XINRU WANG , MINJIAN cHEN and GUANGBO FU Department of Urology, The Affiliated Huai'an No. 1 People's Hospital of Nanjing Medical University, Huai'an, Jiangsu 223300; State Key Laboratory of Reproductive Medicine, Center for Global Health; Key Laboratory of Modern Toxicology of Ministry of Education, School of Public Health, Nanjing Medical University, Nanjing, Jiangsu 211166; Center of Reproduction and Genetic, The Affiliated Huai'an No. 1 People's Hospital of Nanjing Medical University, Huai'an, Jiangsu 223300; Key Laboratory of Environmental Medicine Engineering, Ministry of Education, School of Public Health, Southeast University, Nanjing, Jiangsu 210009; Department of Pathology, The Affiliated Huai'an No. 1 People's Hospital of Nanjing Medical University, Huai'an, Jiangsu 223300; department of Urology, Ningbo Medical Center Lihuili Eastern Hospital, Ningbo, Zhejiang 315040; Department of Pharmacy, The Affiliated Huai'an No. 1 People's Hospital of Nanjing Medical University, Huai'an, Jiangsu 223300, P.R. China Received April 18, 2019; Accepted September 6, 2019 d OI: 10.3892/ijmm.2019.4347 Abstract. Bladder cancer (Bc a) is a common urinary tract resection of bladder tumor (TURBT) may prevent recurrence malignancy with frequent recurrences after initial resection. of urothelial cancer. However, the underlying mechanism Submucosal injection of gemcitabine prior to transurethral remains unknown. In the present study, ultra-performance liquid chromatography Q-Exactive mass spectrometry was used to profile tissue metabolites from 12 BCa patients. The 48 samples included pre- and post-gemcitabine treatment Bc a tissues, as well as adjacent normal tissues. Principal Correspondence to: Professor Guangbo Fu, d epartment of component analysis (Pc A) revealed that the metabolic Urology, The Affiliated Huai'an No. 1 People's Hospital of Nanjing profiles of pre‑gemcitabine BCa tissues differed signic fi antly Medical University, 1 West Huanghe Road, Huai'an, Jiangsu 223300, from those of pre-gemcitabine normal tissues. A total of P.R. c hina E-mail: fgb200@vip.163.com 34 significantly altered metabolites were further analyzed. Pathway analysis using MetaboAnalyst identified three Professor Minjian c hen, State Key Laboratory of Reproductive metabolic pathways closely associated with Bc a, including Medicine, c enter for Global Health, School of Public Health, glutathione, purine and thiamine metabolism, while gluta- Nanjing Medical University, 818 East Tianyuan Road, Nanjing, thione metabolism was also identified by the enrichment Jiangsu 211166, P.R. c hina E-mail: minjianchen@njmu.edu.cn analysis using MetaboAnalyst. In search of the possible targets of gemcitabine, metabolite profiles were compared c ontributed equally between the pre-gemcitabine normal and post-gemcitabine Bc a tissues. Among the 34 metabolites associated with Abbreviations: TURBT, transurethral resection of bladder Bc a, the levels of bilirubin and retinal recovered in Bc a tumor; PCA, principal component analysis; ACN, acetonitrile; tissues treated with gemcitabine. When comparing normal KEGG, Kyoto Encyclopedia of Genes and Genomes; HMDB, bladder tissues with and without gemcitabine treatment, human metabolome database; SMPDB, Small Molecule Pathway among the 34 metabolites associated with Bc a, it was Database; HPLC/MS, high‑performance liquid chromatography/ observed that histamine change may be associated with mass spectrometry; UPLC‑MS, ultra‑performance LC‑MS; the prevention of relapse, whereas thiamine change may be UPLC‑TOF‑MS, UPLC time‑of‑flight MS; UPLC‑HRMS, UPLC involved in possible side effects. Therefore, by employing a high‑resolution MS; BCa, bladder cancer hypothesis-free tissue-based metabolomics study, the present Key words: tissue metabolomics, submucosal injection, gemcitabine, study investigated the metabolic signatures of Bc a and found biomarkers, bladder cancer that bilirubin and retinal may be involved in the mechanism underlying the biomolecular action of submucosal injection of gemcitabine in urothelial Bc a. YANG et al: METABOLOMIc ANALYSIS OF GEM c ITABINE-TREATEd BLA dd ER c ANc ER Introduction Huai'an No. 1 People's Hospital of Nanjing Medical University were recruited between d ecember 2016 and September 2017. Bladder cancer (Bc a) ranks ninth among the most common Bladder tissue samples were collected from the same patient solid tumors worldwide (1). Approximately 75% of newly immediately prior to and 30 min after submucosal injection diagnosed Bc a cases are non-muscle invasive, and the majority of gemcitabine (50 mg, dissolved in 20 ml normal saline). The are histologically low-grade cancer (2). Routine surveillance bladder tissues included Bc a as well as adjacent non-cancerous to monitor Bc a recurrence includes cystoscopic examination bladder tissues. Therefore, a total of 48 samples were collected due to a high risk of recurrence after initial resection. The repeat in four groups: Pre-gemcitabine normal, pre-gemcitabine transurethral resection of bladder tumor (TURBT) remains the Bc a, post-gemcitabine normal, and post-gemcitabine Bc a. first‑line treatment for BCa recurrence (3). However, all these Histopathological diagnosis was conducted by two indepen- invasive procedures result in a high cost of care, and are often dent pathologists according to the classic fi ation criteria of the associated with signic fi ant morbidity. Therefore, more effective World Health Organization/International Society of Urological interventions to prevent Bc a recurrence are urgently needed. Pathology (21). Written informed consent was obtained from Submucosal injection of antitumor drugs (pirarubicin) each participant prior to recruitment. The Ethics c ommittee after standard TURBT was proven to be an effective approach of The Affiliated Huai'an No. 1 People's Hospital of Nanjing to reducing superc fi ial tumor recurrence (4). Gemcitabine is Medical University reviewed and approved the study protocol a pivotal chemotherapeutic agent widely used for Bc a due to (serial no. YL‑P‑2013‑21‑01). The sample size of the present its low toxicity in general (5) and as an intravesical instilla- study was similar to that of a previous study on Pc a tissues, tion (3). d ata from our experimental and clinical studies also which produced valuable findings (22). In addition, the demonstrated that submucosal injection of gemcitabine prior experimental design of self-control and complete collection to TURBT signic fi antly reduced BCa recurrence (6). However, of samples in our study may improve statistical power by the underlying mechanisms are largely unknown. avoiding confounding factors. Metabolomics is a newly emerging technology, which enables the identification of endogenous compounds and Tissue preparation for metabolomic analysis. Following potentially novel mechanisms associated with disease harvesting, all tissues were snap‑frozen in liquid nitrogen, and processes (7). Metabolomics has been used to profile kept in a ‑80˚C freezer until further analysis. The sample prep - metabolites in various biological samples, such as serum (8), aration was conducted as described previously (23). Briey fl , urine (9-15) and tissue (16,17), which are the results of the the tissues were fragmented, ultrasonicated for 5 min (power: metabolic response of living systems to drug toxicity or 60%, pulses: 6/4) in distilled water, and then 150 µl homog- disease (10). Potential biomarkers identified from metabo - enate and 450 µl methanol (Merck KGaA) were mixed in a lomic profiling studies on BCa may be of diagnostic value 1.5-ml Eppendorf tube for protein precipitation. The mixture and act as indicators of cancer recurrence (18). c urrently, a was centrifuged at 16,000 x g for 15 min at 4˚C, and the number of analytical platforms, such as high-performance supernatant was collected and dried in a vacuum centrifugal liquid chromatography/mass spectrometry (HPLc /MS) (10), concentrator. The dry residue was reconstituted in ultra-pure ultra-performance Lc- MS (UPLc- MS) (12), and UPLc water and used for metabolomic analysis. time‑of‑flight MS (UPLC‑TOF‑MS) (15), have been employed to study the metabolomics of Bc a by using urine samples. Metabolomic analysis. The metabolomic analysis was However, metabolomic studies on Bc a tissues is relatively conducted as previously reported (23). Lc- HRMS analysis scarce (16,17). Notably, different metabolomic platforms with was performed on an UPLc Ultimate 3000 system (d ionex), their unique analytical approaches provide complementary coupled with a Q-Exactive mass spectrometer (Thermo Fisher insights into metabolome changes (9,11-15). Therefore, there is Scientic fi , Inc.). The instrument operated at a 70,000 resolution a need to study the tissue-based metabolic signatures of Bc a with a full-scan acquisition ranging from 70 to 1,500 m/z. The using a new metabolomics platform. chromatographic separation of metabolites associated with the Metabolomics has also been applied to cancer treatment metabolomic profiling used a multistep gradient containing and drug target discovery. Eidelman et al reported using ultra‑pure water (mobile phase A) and acetonitrile (ACN; metabolomics to screen the potential therapeutic pathways in mobile phase B), both acidified with 0.1% formic acid. The prostate cancer (Pc a) (19). Metabolomics has also been proven gradient operated at a o fl w rate of 0.4 ml/min over a 15-min to be a promising approach to developing reliable therapeutic period. The metabolites were identie fi d based on the accurate targets for Pc a treatment (20). The present study employed mass and the retention time compared with the commercial liquid chromatography (Lc) -Q-Exactive MS-based metabo- standards. The metabolite standards were purchased from lomic technology to study the metabolic changes in Bc a tissues Sigma‑Aldrich; Merck KGaA, Damas‑beta Co., Ltd., Aladdin before and after treatment with gemcitabine. Identic fi ation of Reagent company and Adamas Reagent co., Ltd. the key metabolites may reveal new metabolic changes associ- ated with Bc a and uncover the changes that mediate the effect Statistical analysis. d ata collected from the mass spec- of gemcitabine in the treatment of Bc a. trometer were processed for pattern recognition analysis (principal component analysis, PCA). Normalized MS data Materials and methods were exported to SIMc A-P+ software (V14.0, Umetrics AB) to perform Pc A where grouping trends could be observed. The Clinical samples. A total of 12 patients (9 men and 3 women; age difference in metabolites between two groups was compared range, 55‑85 years) who had undergone TURBT at the Affiliated by paired t-test. According to previous reports (24,25), the INTERNATIONAL JOURNAL OF MOLEc ULAR MEd Ic INE 44: 1952-1962, 2019 correlation between metabolite changes and cancer stage was (Table IV and Fig. 2B). The metabolic network of the differen- analyzed based on the comparison between two groups using tial metabolites and altered metabolic pathways in the KEGG paired t‑test based on the cancer stage classic fi ation. A P‑value general metabolic pathway map is shown in Fig. 2c . of <0.05 was considered as the threshold for statistically significant differences. Candidate targets of submucosal injection of gemcitabine. To further characterize the metabolic changes and the Based on targeted metabolomic analysis, we next analyzed these metabolic pathways involved, the differentiated metabolites 34 differential metabolite changes in post-gemcitabine Bc a were first annotated with Kyoto Encyclopedia of Genes and tissues, and compared their levels with those in pre-gemcitabine Genomes (KEGG, http://www.genome.jp/kegg/) and Human normal tissues. A total of 32 metabolites maintained signifi - Metabolome d atabase (HMd B, http://www.hmdb.ca/) (date cant changes with identical trends in the comparison between of access for databases: April 19, 2018). d ata were then pre-gemcitabine normal vs. pre-gemcitabine Bc a tissues, processed and analyzed using MetaboAnalyst 4.0 (http://www. indicating that the findings for differential metabolites in metaboanalyst.ca/MetaboAnalyst/) by R software (v3.4.3, Bc a are reliable. Importantly, the significant decrease in GitHub). Two modules of MetaboAnalyst were used, namely the levels of two metabolites associated with Bc a recovered pathway analysis and enrichment analysis, which are based on to insignificant levels following submucosal injection of the KEGG database and Small Molecule Pathway d atabase gemcitabine (Table II and Fig. 3A, B and E-F). These were (SMPd B, http://smpdb.ca/), respectively (26). The metabolic bilirubin and retinal, which may be the candidate targets of network of the differential metabolites and altered metabolic gemcitabine for the prevention of recurrence of urothelial pathways in KEGG general metabolic pathway was visualized BCa. We further analyzed the changes in the two metabolites by iPath 3.0 (http://pathways.embl.de/). in association with cancer stage. In Ta/T1 stage disease, when comparing pre-gemcitabine normal vs. pre-gemcitabine Bc a Results tissues, bilirubin was decreased significantly in the Bc a tissues (P=4.15E-2) (Table SI and Fig. 3c), while this decrease Clinical characteristics of 12 subjects. The mean age of the recovered to an insignificant level following submucosal subjects, including 9 men and 3 women, was 67 years (range, injection of gemcitabine (P=5.40E‑1) (Table SI and Fig. 3D); 55-85 years). In all 12 patients, the diagnosis of urothelial however, at this stage, the decrease of retinal in the tumor was carcinoma was confirmed by histopathological examination. still not statistically signic fi ant (P= 4.76E‑1) (Table SI). In T2 A total of 2 patients had T , 1 patient had T , and 9 patients had stage disease, when comparing pre-gemcitabine normal vs. a 1 T disease. Two patients were diagnosed with high-grade T pre‑gemcitabine BCa tissues, retinal was decreased signifi - 2 2 tumors with squamous metaplasia. The clinical characteristics cantly in the Bc a tissues (P=4.98E-2) (Table SI and Fig. 3G), of the participants are shown in Table I, and are considered to while its change became statistically insignic fi ant following be representative according to the general population of Bc a submucosal injection of gemcitabine (P=3.22E-1) (Table SI in c hina (2,27). and Fig. 3H); however, at this stage, the decrease of retinal in tumor was not statistically signic fi ant (P=1.68E‑1) (Table SI). PCA. A total of 165 metabolites were detected. Pc A was These results indicate that bilirubin and retinal changes were performed to process the metabolite data based on a mean correlated with cancer stage, and gemcitabine may exert its center-scaling model, which is an unsupervised projection effect through metabolic pathways associated with cancer method employed to visually display the intrinsic similari- stage. ties and differences in the dataset. As shown in Fig. 1, Pc A (pre-gemcitabine normal vs. pre-gemcitabine Bc a tissues) Effects of submucosal injection of gemcitabine on normal revealed a well-differentiated and clustered pattern in score tissues. To identify the effects of submucosal injection of plots, indicating the signic fi ant metabolome changes between gemcitabine on normal bladder tissues, the metabolomes of these two groups. normal tissues were compared pre- and post-gemcitabine treat- ment. A total of 10 metabolites were found to be signic fi antly Altered metabolites and cancer‑associated metabolic altered in the normal tissues following submucosal injection of pathways in BCa. UPLC‑Q‑Exactive analysis identified 34 gemcitabine, whereas only 2 metabolites were associated with differentially expressed metabolites annotated in the KEGG Bc a (Table V). Histamine was signic fi antly decreased in BCa, and HMd B databases in pre-gemcitabine Bc a tissues and its level was signic fi antly increased in normal tissues after compared with pre-gemcitabine adjacent normal tissues submucosal injection of gemcitabine (Fig. 3I and J). However, (Table II). thiamine was signic fi antly decreased in BCa, and its level was The 34 Bc a-associated metabolites were then submitted significantly decreased in normal tissues after submucosal to MetaboAnalyst for analysis. Table II lists all metabolites injection of gemcitabine (Fig. 3M and N). We further analyzed found to be altered in Bc a. Table III and Fig. 2A show the the changes in the two metabolite in relation to cancer stage. In metabolic pathways connected with these 34 metabolites, Ta/T1 stage disease, when comparing pre-gemcitabine normal among which three pathways, namely glutathione, purine vs. pre-gemcitabine Bc a tissues, the changes in histamine and thiamine metabolism, were signic fi antly associated with (P=2.60E-1) and thiamine (P=8.39E-2) in the tumor were Bc a. Furthermore, in order to expand our understanding of insignic fi ant (Table SI). However, in T2 stage disease, when metabolic pathways related to Bc a, the module of enrich- comparing pre-gemcitabine normal vs. pre-gemcitabine Bc a ment analysis of MetaboAnalyst was used, which verie fi d that tissues, the decrease in histamine (P=4.61E-3) (Table SI and glutathione metabolism was signic fi antly associated with BCa Fig. 3K) and thiamine (P=4.13E-4) (Table SI and Fig. 3O) in YANG et al: METABOLOMIc ANALYSIS OF GEM c ITABINE-TREATEd BLA dd ER c ANc ER Table I. clinical characteristics of 12 Bca patients. Histopathology --------------------------------------------------------------------------- Number Sex Age (years) Tumor type Stage Grade 01 Male 55 MIUc T2 High 02 Male 56 MIUc T2 High 03 Male 59 MIUc T2 High 04 Female 82 MIUc T1 High 05 Male 67 MIUc T2 High 06 Male 76 MIUc T2b Squamous metaplasia 07 Male 76 MIUc T2 High 08 Female 60 MIUc T2 High 09 Male 67 NMIUc Ta High 10 Male 58 NMIUc Ta High 11 Male 85 MIUc T2a High 12 Female 61 MIUc T2 Squamous metaplasia MIUC, muscle‑invasive urothelial carcinoma; NMIUC, non‑muscle‑invasive urothelial carcinoma. BCa, bladder cancer. Figure 1. Pc A score plots derived from pre-gemcitabine normal (green) and pre-gemcitabine Bc a (blue) tissues. c omparison of pre-gemcitabine normal (green) and pre‑gemcitabine BCa (blue) tissues. PCA, principal component analysis; BCa, bladder cancer. the BCa tissues was signic fi ant. In T2 stage disease, the hista - (P=4.11E-2) (Table SI and Fig. 3P). These results indicate that mine level was signic fi antly increased in normal tissues after these metabolite changes were correlated with cancer stage, submucosal injection of gemcitabine (P=3.55E-3) (Table SI and gemcitabine may exert its effects mainly through these and Fig. 3L), while thiamine was significantly decreased metabolic pathways in T2 stage disease. INTERNATIONAL JOURNAL OF MOLEc ULAR MEd Ic INE 44: 1952-1962, 2019 Table II. List of the altered metabolites identified in BCa and their changes in the comparison between BCa tissues with or without gemcitabine pretreatment and pre-gemcitabine normal tissues. Post-gemcitabine Bca Pre-gemcitabine Bca vs. pre-gemcitabine a a vs. normal tissues normal tissues -------------------------------------------------- ------------------------------------------------- a a Metabolites KEGG HMdB Fold change P-value Fold change P-value deoxycytidine c00881 HMdB0000014 4.03 1.22E-02 10.10 4.15E-05 5'‑Methylthioadenosine C00170 HMDB0001173 15.16 2.38E‑04 12.22 4.16E‑04 3'‑AMP C01367 HMDB0003540 4.95 3.02E‑02 6.36 3.33E‑02 Androstenedione c00280 HMdB0000053 0.16 4.09E-02 0.10 5.76E-03 Bilirubin C00486 HMDB0000054 0.4 3.97E‑02 1.26 2.04E‑01 cholic acid c00695 HMdB0000619 0.29 3.14E-02 0.24 2.00E-03 cytidine c00475 HMdB0000089 3.83 2.43E-03 3.05 6.77E-03 5-Hydroxylysine c16741 HMdB0000450 0.64 4.40E-02 0.48 7.49E-03 deoxyinosine c05512 HMdB0000071 2.44 3.87E-02 4.12 1.70E-02 Glucosamine 6-phosphate c00352 HMdB0001254 6.04 6.26E-03 6.87 3.02E-05 Glyceraldehyde c02154 HMdB0001051 0.29 5.86E-04 0.32 1.65E-03 Sphingosine c00319 HMdB0000252 2.03 3.30E-02 3.34 2.18E-05 Glycerophosphocholine c00670 HMdB0000086 56.88 1.93E-03 51.03 3.44E-03 Glycine c00037 HMdB0000123 0.55 2.22E-02 0.47 4.68E-03 Guanidine c17349 HMdB0001842 0.47 5.25E-03 0.01 3.79E-04 Hexadecanedioic acid c19615 HMdB0000672 0.27 1.34E-02 0.23 3.44E-03 Histamine c00388 HMdB0000870 0.58 2.49E-02 0.35 7.49E-03 Hypotaurine c00519 HMdB0000965 0.07 4.19E-04 0.08 5.33E-04 Inosinic acid c00130 HMdB0000175 92.72 1.73E-05 150.57 1.43E-03 L-carnitine c00318 HMdB0000062 2.24 2.39E-03 2.70 3.97E-03 L-cystine c00491 HMdB0000192 0.49 2.70E-02 0.13 1.17E-03 L-Phenylalanine c00079 HMdB0000159 0.51 2.80E-02 0.31 4.70E-03 N-Acetylneuraminic acid c19910 HMdB0000230 2.68 2.13E-02 2.90 3.38E-02 Oxidized glutathione C00127 HMDB0003337 13.97 4.36E‑03 16.05 2.77E‑03 L-Palmitoylcarnitine c02990 HMdB0000222 3.59 1.88E-02 5.09 2.77E-04 Pantothenol c05944 HMdB0004231 0.44 3.12E-02 0.18 8.44E-05 Pyroglutamic acid c01879 HMdB0000267 0.29 1.85E-03 0.16 5.30E-04 Quinic acid c06746 HMdB0003072 0.09 4.05E-02 0.25 4.76E-02 Retinal C00376 HMDB0001358 0.13 4.98E‑02 0.43 3.81E‑01 Rhamnose c00507 HMdB0000849 0.22 2.90E-03 0.17 2.40E-04 deoxycholic acid glycine conjugate c05464 HMdB0000631 0.19 6.50E-03 0.15 4.52E-04 Sorbitol c00794 HMdB0000247 0.29 9.19E-03 0.13 1.44E-03 Tetradecanedioic acid c11002 HMdB0000872 0.44 4.46E-02 0.36 4.47E-02 Thiamine c00378 HMdB0000235 0.34 6.95E-05 0.18 2.47E-05 In the fold change calculation, the metabolite in pre-gemcitabine normal tissues served as the denominator. Bold print indicates statistical significance. BCa, bladder cancer; KEGG, Kyoto Encyclopedia of Genes and Genomes; HMDB, human metabolome database. Discussion glutathione metabolism was the consistently altered pathway in enrichment and pathway analyses (Tables III and IV, and In the present study, UPLc- Q-Exactive-based metabolomic Fig. 2A and B); iii) among the 34 cancer‑associated metabolites, analysis was utilized to profile metabolites in BCa tissues. the levels of bilirubin and retinal recovered after gemcitabine The major findings may be summarized as follows: i) A total injection, suggesting that these two are likely the targets of of 34 key metabolites associated with BCa were identified gemcitabine treatment (Table II, Fig. 3A‑D and E‑H); iv) the (Table II); ii) three metabolic pathways, namely glutathione, effects of gemcitabine on normal bladder tissues were also purine and thiamine metabolism, were altered in Bc a, and investigated, and it was deduced that histamine may have the YANG et al: METABOLOMIc ANALYSIS OF GEM c ITABINE-TREATEd BLA dd ER c ANc ER Table III. Pathway analysis of metabolite changes in Bca . KEGG pathway Total Hits P-value Glutathione metabolism 38 3 1.55E-02 Purine metabolism 92 4 3.86E-02 Thiamine metabolism 24 2 4.40E-02 Nitrogen metabolism 39 2 1.04E-01 Primary bile acid biosynthesis 47 2 1.41E-01 Fructose and mannose metabolism 48 2 1.46E-01 cysteine and methionine metabolism 56 2 1.86E-01 cyanoamino acid metabolism 16 1 2.04E-01 Pyrimidine metabolism 60 2 2.07E-01 Taurine and hypotaurine metabolism 20 1 2.48E-01 Retinol metabolism 22 1 2.70E-01 Ether lipid metabolism 23 1 2.80E-01 Aminoacyl-tRNA biosynthesis 75 2 2.86E-01 Alanine, aspartate and glutamate metabolism 24 1 2.90E-01 Sphingolipid metabolism 25 1 3.01E-01 Pantothenate and coA biosynthesis 27 1 3.20E-01 Phenylalanine, tyrosine and tryptophan biosynthesis 27 1 3.20E-01 Methane metabolism 34 1 3.86E-01 Glycerophospholipid metabolism 39 1 4.28E-01 Porphyrin and chlorophyll metabolism 104 2 4.36E-01 Galactose metabolism 41 1 4.45E-01 Histidine metabolism 44 1 4.68E-01 Phenylalanine metabolism 45 1 4.76E-01 Lysine degradation 47 1 4.91E-01 Glycine, serine and threonine metabolism 48 1 4.98E-01 Fatty acid metabolism 50 1 5.13E-01 Amino sugar and nucleotide sugar metabolism 88 1 7.21E-01 Steroid hormone biosynthesis 99 1 7.63E-01 The analysis was conducted by the module of pathway analysis of MetaboAnalyst 4.0. BCa, bladder cancer; KEGG, Kyoto Encyclopedia of Genes and Genomes. ability to protect against disease recurrence, whereas thiamine altered oxidized glutathione in BCa; they discovered four may be involved in the side effects of treatment (Table V, single-nucleotide polymorphisms in the glutathione synthetase Fig. 3I‑L and M‑P), which requires further confirmation in gene, and these changes were associated with Bc a recurrence future studies. after TUR and Bacillus c almette Guerin treatment (31). Our The identic fi ation of the three metabolic pathways signifi - findings, together with others, suggest that oxidative stress in cantly altered in Bc a may be pathophysiologically important. Bc a cells is at least partially due to the disrupted glutathione Glycine is an important amino acid that participates in all metabolism. three metabolic pathways. A decrease in glycine was reported By using Lc- Q-Exactive MS, this study is, to the best of as a biomarker in Bc a (14). As a precursor of purine synthesis, our knowledge, the first to investigate the metabolite changes reduced glycine in Bc a indicates that a critical metabolic in Bc a treated with gemcitabine followed by TURBT. Among process associated with cell proliferation is altered in Bc a (14). the 34 cancer-associated metabolites, the levels of bilirubin and Notably, glutathione metabolism is the consistently altered retinal recovered after gemcitabine treatment, indicating that pathway in enrichment and pathway analysis. Glutathione they may be the potential targets of gemcitabine for reducing is the most abundant low-molecular-weight peptide present Bc a recurrence. Bilirubin, a degradation product of free heme in eukaryotic cells (28). Glutathione is a primary cellular groups, protected LLc- PK1 cells against cisplatin-induced antioxidant that effectively scavenges free radicals and other death (32). A large population-based study demonstrated reactive oxygen species (29) and, therefore, plays an important that patients with primary biliary cirrhosis (PBc) have a role in protecting cells from oxidative injury (30). Glutathione nine-fold increased risk of developing urinary Bc a, and Bc a is also involved in cellular detoxic fi ation, and is required in and PBc share a number of etiological factors (33). Uridine several aspects of the immune response (31). Ke et al reported 5'‑diphospho‑glucuronosyltransferases (UGTs) are enzymes INTERNATIONAL JOURNAL OF MOLEc ULAR MEd Ic INE 44: 1952-1962, 2019 Figure 2. Results of the metabolic connection analysis of the changed metabolomic data in Bc a. (A) Pathway analysis based on the KEGG database. (B) Enrichment analysis based on SMPd B. (c ) Metabolic network of the differential metabolites and altered metabolic pathways in KEGG general metabolic pathway map. Red dots represent the increased metabolites in BCa; green dots represent the specic fi ally decreased metabolites in BCa; orange line, glutathione metabolism; purple line, purine metabolism; blue line, thiamine metabolism. The metabolites and pathways not indicated in the general pathway map are not shown. The original general metabolic pathway map is available at https://pathways.embl.de/ipath3.cgi. KEGG, Kyoto Encyclopedia of Genes and Genomes; SMPDB, Small Molecule Pathway Database; BCa, bladder cancer. that participate in several biological processes involving Bc a (37). A high dietary intake of vitamin A reduces the bilirubin conjugation. UGTs catabolize carcinogens and, there- incidence of BCa (38). Ziouzenkova et al reported retinal as fore, protect bladder cells from the harmful effects of toxic a distinct biological regulator involved in suppressing adipo- chemicals accumulated in the bladder. Targeting UGT1A may genesis, diet-induced obesity and insulin resistance (39). The serve as a novel therapeutic intervention against uroepithelial potential effect of bilirubin and retinal on the clinical outcome carcinomas (34). Retinal (retinaldehyde; Rald) is derived from of patients with BCa identie fi d in the present study is worthy retinol (vitamin A) oxidized by alcohol dehydrogenases (35). of further investigation in the future. Retinal plays an essential role in molecular signaling in The effects of gemcitabine on the metabolism of adjacent vision, and serves mainly as a retinoic acid (an active form of normal tissues were also examined. Most gemcitabine-induced vitamin A) precursor outside the eye (36). The serum levels metabolites do not overlap with those identie fi d in BCa, indi - of vitamin A were found to be decreased in patients with cating that gemcitabine did not exert notable adverse effects on YANG et al: METABOLOMIc ANALYSIS OF GEM c ITABINE-TREATEd BLA dd ER c ANc ER Table IV. Enrichment analysis of metabolite changes in Bca . Pathway from SMPdB Total Hits P-value Glutathione metabolism 21 3 2.96E-02 carnitine synthesis 22 2 1.64E-01 Bile acid biosynthesis 65 4 1.64E-01 Glutamate metabolism 49 3 2.19E-01 Thiamine metabolism 9 1 2.63E-01 Fructose and mannose degradation 32 2 2.88E-01 Amino Sugar metabolism 33 2 3.00E-01 Pyrimidine metabolism 59 3 3.11E-01 Taurine and hypotaurine metabolism 12 1 3.35E-01 Retinol metabolism 37 2 3.50E-01 Porphyrin metabolism 40 2 3.87E-01 Fatty acid metabolism 43 2 4.23E-01 Methionine metabolism 43 2 4.23E-01 Alanine metabolism 17 1 4.39E-01 Beta oxidation of very long-chain fatty acids 17 1 4.39E-01 Purine metabolism 74 3 4.51E-01 Spermidine and spermine biosynthesis 18 1 4.58E-01 Pantothenate and coA biosynthesis 21 1 5.11E-01 Androstenedione metabolism 24 1 5.59E-01 Glycerolipid metabolism 25 1 5.74E-01 Oxidation of branched chain fatty acids 26 1 5.89E-01 Mitochondrial beta-oxidation of short-chain saturated fatty acids 27 1 6.03E-01 Mitochondrial beta-oxidation of long-chain saturated fatty acids 28 1 6.16E-01 Phenylalanine and tyrosine metabolism 28 1 6.16E-01 Ammonia recycling 32 1 6.66E-01 Androgen and estrogen metabolism 33 1 6.78E-01 Aspartate metabolism 35 1 6.99E-01 Galactose metabolism 38 1 7.29E-01 Sphingolipid metabolism 40 1 7.48E-01 Histidine metabolism 43 1 7.73E-01 Arginine and proline metabolism 53 1 8.41E-01 Glycine and serine metabolism 59 1 8.72E-01 Tryptophan metabolism 60 1 8.76E-01 Arachidonic acid metabolism 69 1 9.10E-01 The analysis was conducted by the module of enrichment analysis of MetaboAnalyst 4.0. Bca, bladder cancer. SMPdB, Small Molecule Pathway database. normal bladder tissues. It was observed that histamine change was signic fi antly higher compared with that in rats fed bracken may be associated with the prevention of relapse. Histamine fern but receiving no thiamine supplements, as thiamine may is derived from the decarboxylation of histidine by histidine interfere with the absorption, distribution, metabolism, or decarboxylase in mammals (8). Histamine is primarily released excretion of the bracken fern (43). However, a case-control in ina fl mmatory processes by mast cells (8), which are closely study from New Hampshire investigated the effect of minerals associated with Bc a (40). Histamine H1 receptor (HRH1) and vitamins on the risk of Bc a, and found that a higher total expression was identie fi d in BCa and found to be associated intake of thiamine was inversely correlated with Bc a risk in with the prognosis (41). It was observed that thiamine change older participants (44). may be involved in treatment-related side effects. Thiamine, There is currently a lack of effective biomarkers for Bc a or vitamin B-1, is a water-soluble vitamin (42). An early report diagnosis and prognosis. Metabolomic profiles from tissue by Pamukcu et al demonstrated that the incidence of urinary have the potential to be used, along with other current diag- bladder carcinomas in rats fed bracken fern and additionally nostics, to help guide the clinical management of patients s.c. injected once weekly with 2 mg of thiamine hydrochloride with BCa. The changed metabolites identie fi d in the present INTERNATIONAL JOURNAL OF MOLEc ULAR MEd Ic INE 44: 1952-1962, 2019 Table V. changes in normal tissues after gemcitabine treatment. Pre-gemcitabine Post-gemcitabine vs. a a Bca vs. normal tissues pre-gemcitabine normal tissues ------------------------------------------------------------------- ------------------------------------------------------------------- a a Metabolites Fold-change P-value Fold-change P-value 3-Methyladenine 1.54 3.78E-01 0.17 2.33E-02 Ascorbic acid 22.26 1.85E-01 0.28 5.46E-03 creatinine 0.48 3.44E-01 0.13 1.16E-02 d-Glyceraldehyde 3-phosphate 0.72 3.33E-01 0.15 2.16E-02 Histamine 0.58 2.49E-02 2.26 3.73E-04 N-Acetylglutamic acid 0.52 8.58E-02 0.57 4.95E-02 N-Acetylglutamine 0.68 5.45E-01 0.11 4.25E-02 Thiamine 0.34 6.95E-05 0.65 2.11E-02 Tryptamine 0.10 7.66E-02 0.11 3.23E-02 Uridine 1.41 6.74E-02 1.51 3.65E-02 In the fold change calculation, the metabolite in pre-gemcitabine normal tissues served as the denominator. Bold print indicates statistically significant differences. BCa, bladder cancer. Figure 3. Relative levels of potential candidate targets of submucosal injection of gemcitabine (A, B, E, F, I, J, M and N). All samples; (C and D) Ta/T1 stage; (G, H, K, L, O and P) T2 stage. Values are presented as mean ± standard error of the mean. The P-values of paired t-test are indicated. YANG et al: METABOLOMIc ANALYSIS OF GEM c ITABINE-TREATEd BLA dd ER c ANc ER study, such as glycine, may be used as potential biomarkers Patient consent for publication for Bc a. In addition, those metabolites discovered in Bc a with gemcitabine treatment may reveal new metabolic pathways Not applicable. that mediate the anti-recurrence effect of gemcitabine, which currently remain elusive. Competing interests In summary, the present study employed UPLc- HRMS- based metabolomic analysis to investigate metabolite changes The authors declare that they have no competing interests. in bladder tissues from Bc a and Bc a treated with gemcitabine. The findings may provide new insights into metabolic changes Authors' information in Bc a and the biomolecular basis of submucosal injection of gemcitabine for Bc a. In addition, the study demonstrated Chao Yang: 15949177271@163.com; Xian Sun: 1019949991@ that the UPLc- HRMS-based metabolomic analysis provides qq.com; Hengbing Wang: wanghengbing2004@163.com; comprehensive metabolite profiling data that may pave the Ting Lu: 552095014@qq.com; Keqing Wu: wkwly1993@ way to a novel approach to Bc a research. qq.com; Yusheng Guan: guanys@njmu.edu.cn; Jing Tang: tjing19681222@sina.com; Jian Liang: lj3936@126.com; Acknowledgements Rongl i Su n: 101012172@seu. e du.cn; Z hong yi ng Guo: guozhongying407@163.com; Sinian Zheng: zhengsinian@163. Not applicable. com; Xiaoli Wu: wuxiaoli2233@126.com; Hesong Jiang: njjhs2007@163.com; Xi Jiang: piscesjxi@163.com; Bing Funding Zhong: 15152569186@163.com; Xiaobing Niu: bingke2008@ si na.com; Sua n Sun: hay yssa@163.com; X i n r u Wa ng: The present study was supported by grants from the National xrwang@njmu.edu.cn Natural Science Foundation (81872650 and 81573182); the Natural Science Foundation of the Jiangsu Higher Education References Institutions of China (18KJA320003 and 18KJB320001); 1. Ferlay J, Soerjomataram I, d ikshit R, Eser S, Mathers c , the Key Research & d evelopment Plan of Jiangsu Province Rebelo M, Parkin d M, Forman d and Bray F : c ancer incidence (BE2017628); the Southeast University & Nanjing Medical and mortality worldwide: Sources, methods and major patterns University Collaborative Research Project (2242018K3DN25); in GLOBOc AN 2012. Int J c ancer 136: E359-E386, 2015. 2. 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International Journal of Molecular Medicine – Pubmed Central

Published: Sep 23, 2019

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