OR Oncology Reports 1021-335X 1791-2431 D.A. Spandidos 10.3892/or.2018.6220 or-39-03-1261 Articles The role of aldehyde dehydrogenase 1A1 in B-cell non-Hodgkin's lymphoma SongYing-Hui 1 ZhongMei-Zuo 2 ChaiQin 1 GongKui-Yu 1 TanXiao-Lang 1 HuJin-Yue 3 WangGui-Hua 1 Department of Oncology, Changsha Central Hospital, Hunan 410004, P.R. China Department of Oncology, Xiangya Hospital, Central South University, Changsha, P.R. China Central Laboratory, Changsha Central Hospital, Changsha, P.R. China Correspondence to: Professor Gui-Hua Wang, Department of Oncology, Changsha Central Hospital, Changsha, Hunan 410004, P.R. China, E-mail: doctorwang_031@163.com 032018 18012018 39 3 1261 1268 30072017 11012018 Copyright © 2018, Spandidos Publications 2018

Previously we showed that aldehyde dehydrogenase 1A1 (ALDH1A1) is a new mediator for resistance of DLBCL to CHOP and a facility predictor of clinical prognosis. In the present study, knockdown and inhibitor of ALDH1A1 were applied to identify the role of ALDH1A1 in Raji cells. CCK-8 and clone formation assay were applied to determine the CHOP sensitivity and clone formation ability. Caspase colorimetric assay and Annexin V/FITC staining was performed to determine the degree of apoptosis. Western blot analysis was used to detect the NF-κB/STAT3 signaling proteins and apoptotic-associated proteins. Real-time quantitative PCR (RT-PCR) was used to identify the differential expression of ALDH1A1 between NHL patients and healthy donors. We demonstrated that inhibition of ALDH1A1 increased the sensitivity of Raji cells to CHOP, as indicated by increased cytotoxicity, reduced clonogenicity, activated caspase-3/-9, decreased NF-κB/STAT3 signaling and increased pro-apoptosis signaling, ad increased apoptosis rate. Moreover, we found high ALDH1A1 expression was associated with poor prognosis in NHL patients. Our data revealed the critical role of ALDH1A1 in NHL and provides a theoretical basis for the use of ALDH1A1 inhibitors in NHL patients.

 B-cell non-Hodgkin's lymphoma ALDH1A1 drug resistance prognostication Introduction

Non-Hodgkin's lymphoma (NHL) consists of many histologically and biologically unique lymphoid malignancies (1). In Western countries, 85% of NHL is of B-cell origin (2). Unfortunately, little progress has been made in improving the survival of NHL patients receiving standard therapy, largely due to insensitivity or resistance of the cancer cells to treatment. Many patients have short complete remission or early relapse after treatment, which shortens survival and causes tremendous psychological and physical pain.

Recently, in vitro studies have shown that chemotherapy response depends on activation of the apoptosis cascade (3). Moreover, the most important factor in chemotherapy resistance is suppression of the apoptosis pathway, which leads to disease recurrence in NHL patients. Imbalances in apoptosis regulation are associated with the abnormal activation of growth signal transduction pathways, and constitutive activation of these pathways, including NF-κB and STAT3, has been shown to occur in NHL tissue and cell lines. Activation of these pathways is thought to be the major cause of cancer cell resistance to chemotherapeutics via downstream alterations in apoptosis pathway regulation (4,5).

Accumulating data suggest that aldehyde dehydrogenase 1A1 (ALDH1A1) is also involved in the chemotherapy resistance of tumor cells (6–8). ALDH1A1 is overexpressed in a variety of solid tumors and leukemias, and it is a newly discovered cancer stem cell (CSC) marker (9–11). Hodgkin's lymphoma cells with high expression of ALDH1A1 possess the characteristics of stem cells (12). Notably, p-STAT3 plays a role in maintaining CSC characteristics in colon cancer (13), whereas NF-κB plays a similar role in pancreatic cancer (14). Recently, we demonstrated that knockdown or inhibition of ALDH1A1 increases chemosensitivity in diffuse large B-cell lymphoma (DLBCL) Farage cells, potentially via modulation of NF-κB/STAT3 signaling (15); however, in contrast, Fujita et al demonstrated by immunohistochemistry that ALDH1 is not expressed in DLBCL (16). Subsequently, we found that ALDH1A1 confers chemoresistance in DLBCL Pfeiffer cells, and that its expression is associated with poor prognosis in DLBCL patients (17).

In the present study, we analyzed ALDH1A1 expression in human NHL patient samples, and we assessed the relationship between ALDH1A1 expression and B-cell NHL patient prognosis. Furthermore, we choose the Raji cell line, a Burkitt's lymphoma cell line as a model since Raji cells with mutant p53, constitutively activated NF-κB and increased BCL-2 expression which are commonly present in patients with NHL and are considered a source of chemotherapy failure in patients whose disease are chemoresistant in B-cell NHL (18). we used the Raji cell line to explore the role of ALDH1A1 in chemotherapy resistance, via modulation of NF-κB/STAT3 signaling and apoptosis, in B-cell NHL.

 Patients and methods <sec> <title>Patient characteristics

The samples were obtained from 112 patients treated in the Xiang-Ya Hospital of Central South University (Hunan, China) after being diagnosed with B-cell NHL according to the WHO (2008) classification, and was confirmed by pathological histology, from 2013 to 2014. Indolent lymphoma defined follicular lymphoma, marginal zone lymphoma, mucosa associated lymphoid tissue type, unclassified small B cell lymphoma. Progressive lymphoma contained diffuse large B cells, mantle cells and Burkitt's lymphoma. For comparison, we obtained samples from 24 healthy donors as the normal controls. All patients were enrolled following approval from the Ethics Committee, and they all provided informed consent. From each patient, 3- to 5-ml peripheral blood samples were collected in sterile tubes containing anticoagulant (heparin sodium) before they received treatment. Mononuclear cells (MNCs) were enriched by density centrifugation over Ficoll-Paque (TBD Science, Tianjin, China) and stored at −80°C.

 RNA isolation and real-time PCR

Cells were lysed, and the total RNA was extracted with TRIzol reagent (Invitrogen, Carlsbad, CA, USA) according to the manufacturer's instructions. Τotal RNA (1 µg) was used in cDNA synthesis. Reverse transcription of RNA was carried out with a PrimeScript™ RT reagent kit (Takara Bio, Inc., Otsu, Japan). Synthesized cDNA was subjected to quantitative real-time (qRT)-PCR for the detection of ALDH1A1 and GAPDH using the SYBR-Green fluorescence-based Assay kit (Takara). The following primers were used: 5′-TGTTAGCTGATGCCGACTTG-3′ and 5′-TTCTTAGCCCGCTCAACACT-3′ for ALDH1A1; and 5′-ACCACAGTCCATGCCATCAC-3′ and 5′-TCCACCACCCTGTTGCTGTA-3′ for GAPDH (Sangon Biotech, Shanghai, China). The following amplification conditions were used: pre-denaturation at 95°C for 30 sec, 40 cycles of denaturation at 95°C for 5 sec, annealing at 60°C for 34 sec, and elongation at 72°C for 60 sec, and a final extension at 72°C for 10 min. The relative quantification (RQ) of ALDH1A1 was calculated based on the threshold cycle (Ct) values as follows: RQ = 2−ΔΔCt, where ΔΔCt = [Ct(ALDH1A1) - Ct(GAPDH)] sample (patients) - [Ct (ALDH1A1) - Ct(GAPDH)] sample (healthy controls). The qRT-PCR was performed using the ABI 7500 Fast Real-Time PCR system (Applied Biosystems, Foster City, CA, USA).

 Cell culture

Raji cells [human Burkitt's lymphoma cell line, ATCC CCL-86) (ATCC; American Type Culture Collection, Manassas, VA, USA)] were grown in RPMI-1640 medium containing 10% fetal bovine serum (FBS) and 100 U/ml penicillin/streptomycin and incubated at 37°C in 5% CO2.

 Chemical treatment and cell survival rate assay

Cells (5,000) were seeded on a 96-well plate in a volume of 100 µl. CHOP treatment was performed using cyclophosphamide, vincristine, adriamycin and prednisone at a clinical ratio of 80/5.5/0.16/11.1, respectively, with dosages ranging from 5–1,280 ng/ml (19). Cytotoxicity was assessed using the Cell Counting Kit-8 (CCK-8) assay according to the manufacturer's instructions (Dojindo, Kumamoto, Japan). Forty-eight hours after drug treatment, CCK-8 was added to each well, and the OD at 450 nm of each sample was determined using a microplate spectrophotometer (Bio-Rad, Hercules, CA, USA).

 Colony formation assay

Cells were seeded into 6-well culture plates at a density of 1,000 cells/well in triplicate, with 2 ml of methylcellulose (stem cell) mixture containing Dulbecco's modified Eagle's medium (DMEM) and FBS supplemented with 10% fetal bovine serum (FBS), with or without 400 ng/ml CHOP. The numbers of colonies were counted on day 14.

 ALDH1A1 knockdown by shRNA

Lentiviral vectors expressing short hairpin RNAs (shRNA) against ALDH1A1 (GenBank accession no. NM_000689) were obtained from GeneChem Co., Ltd. (Shanghai, China), and synthesized with the following strand sequences: forward, 5′-tcgGGCTAAGAAGTATATCCTTctcgagAAGGATATACTTCTTAGCCcgttttttc-3′ and reverse, 5′-TCGAGAAAAAAcgGGCTAAGAAGTATATCCTTCTCGAGAAGGATATACTTCTTAGCCCGA-3′. The lentivirus was transfected according to the Lentiviral Vector Particle operation manual instructions as previously described (15). Validation of the knockdown was performed at the protein level by western blotting, and at the messenger RNA (mRNA) level by relative qRT-PCR.

 Detection of active caspase-3 and −9

Caspase activity was assayed using the Caspase Colorimetric Assay kit (KeyGen Biotech, Nanjing, China) according to the manufacturer's protocol. Briefly, cells were harvested and lysed for 30 min. Then, 50 µl samples were mixed with reaction buffer and the caspase-3/-9 substrate and incubated for 4 h at 37°C in the dark. The percentage of A405 values for the test samples vs. those for the control samples indicated the percentage of caspase activity.

 Flow cytometric assay for apoptosis

Cell apoptosis was assayed using the Annexin V/FITC apoptosis detection kit (Beijing Biosea Biotechnology Co., Ltd., Beijing, China) according to the manufacturer's protocol. Data acquisition and analysis were performed using a flow cytometer (Becton-Dickinson, Franklin Lakes, NJ, USA).

 Western blotting

Western blotting was carried out as previously described (15). Total protein (20 µg) was loaded per well. The following antibodies and dilutions were used: anti-ALDH1A1 (1:500; Abcam, Cambridge, MA, USA), anti-NF-κB (1:1,000), anti-p-NF-κB (1:1,000), anti-STAT3 (1:1,000), anti-p-STAT3 (1:1,000), anti-BCL-2 (1:1,000), anti-BAX (1:1,000), anti-caspase-3 (1:1,000), anti-caspase-9 (1:1,000) (all from Cell Signaling Technology, Inc., Danvers, MA, USA). Anti-GAPDH (1:2,000; Goodhere Biotechnology Co., Ltd., Hangzhou, China) served as a loading control.

 Statistical analysis

All data shown represent the results of at least three independent experiments. The calculations were analyzed using the Statistical Package for the Social Sciences (SPSS) software. For analysis of the survival data, the Kaplan-Meier model was applied, and the log-rank test was performed. The association between ALDH1A1 expression and clinicopathological features was studied using the χ2 test. Differences between the results of experimental treatments and the average cloning number were assessed by one-way analysis of variance. Differences were two-tailed and considered significant at values of P<0.05. The diagrams were generated using GraphPad Prism 5 software (GraphPad Software, Inc., La Jolla, CA, USA).

 Results <sec> <title>High levels of ALDH1A1 expression are associated with an unfavorable prognosis in NHL patients

Quantitative real-time (qRT)-PCR analysis of ALDH1A1 expression was conducted in peripheral blood samples from 112 NHL and 24 healthy control patients. The median relative quantification (RQ) values of ALDH1A1 in NHL and control patients were 0.326 (range, 0.010–5.918) and 0.041 (range, 0.010–0.492), respectively; thus, ALDH1A1 levels were significantly higher in NHL patients than in controls (P<0.05; Fig. 1A).

Next, we used the median RQ value of ALDH1A1 to separate the patients into a high ALDH1A1 group (>0.326) or a low ALDH1A1 group (<0.326). The 56 patients with high expression had a median ALDH1A1 RQ of 0.846 (range, 0.336–5.92), whereas those with low expression had a median ALDH1A1 RQ of 0.148 (range, 0.010–0.316). Baseline ALDH1A1 levels were correlated with patient lactate dehydrogenase (LDH) levels (P=0.014), performance status (PS) (P=0.011), Ann Arbor stage (P>0.05), International Prognostic Index (IPI) score (P>0.05), and lymphoma category (P=0.001), but not with other factors (Table I). Importantly, patients in the high ALDH1A1 group showed shorter cumulative survival than those in the low ALDH1A1 group (P<0.0001; Fig. 1B). The expression of ALDHA1 in indolent lymphoma was significantly lower than in progressive lymphoma. Although in the experimental group of 42 cases of indolent lymphoma patient's data showed that ALDH1A1 difference in expression related to difference cumulative survival rate (P=0.031), but in multivariate analysis ALDH1A1 was not an independent prognostic indicator (P=0.053). The number of indolent lymphoma patients was too small to get a convinced conclusion. Whether ALDH1A1 was also suitable for inactive lymphoma is unknown and required more evidence to validate it. Moreover, among all NHL patients, IPI score, lymphoma category and ALDH1A1 levels were independent prognostic indicators (Table II).

 Inhibition of ALDH1A1 resensitizes Raji cells to the CHOP regimen

Next, we performed western blot analysis and showed that ALDH1A1 expression was higher in human Burkitt's lymphoma Raji cells than in human B-cells (negative control) (Fig. 2A and B). To determine whether ALDH1A1 mediates resistance to CHOP chemotherapy in Raji cells, the ALDH1A1 inhibitor DEAB was applied to Raji cells in combination with CHOP treatment, and the resulting cytotoxicity was assessed. The cytotoxicity in the combination group (CHOP plus DEAB) was higher than in the CHOP group alone at each concentration (Fig. 2C). Moreover, the IC50 values of the Raji cells to the CHOP regimen decreased from 685±25.62 to 412.56±38.58 ng/ml (P=0.013) in the presence of DEAB (Fig. 2C).

 Knockdown or inhibition of ALDH1A1 reduces clonogenic capacity and increases apoptotic activity in Raji cells

To further determine the mechanisms of ALDH1A1 action, we performed loss-of-function studies, via ALDH1A1 inhibition or knockdown, in Raji cells. First, we confirmed successful shRNA-mediated knockdown of ALDH1A1 by western blot analyses (Fig. 2A and B). In colony formation assays, the numbers of colonies in the DEAB treatment group was significantly less than those of the Raji-wt control group, both in the absence or presence of CHOP treatment (Fig. 3A). Similarly, the numbers of colonies in the Raji-shRNA group was significantly less than those in the Raji-mock group, both in the absence or presence of CHOP treatment (Fig. 3B). These data demonstrated that ALDH1A1 loss-of-function reduced the clonogenic capacity of Raji cells.

Regarding apoptotic effects, colorimetric caspase assays showed that caspase-3 activity was increased in the DEAB and shRNA groups compared with those of their respective control groups, both in the absence or presence of CHOP treatment (Fig. 3C). Consistent with these data, caspase-9 activity was increased in the DEAB and shRNA groups compared with those of their respective control groups, both in the absence or presence of CHOP treatment (Fig. 3D). Finally, Annexin V and PI staining revealed an increased apoptosis rate in the shRNA group compared those of the wt or mock groups (Fig. 3E). Moreover, the shRNA group showed an even greater apoptosis rate after 400 ng/ml CHOP treatment for 24 h (Fig. 3E).

 Knockdown or inhibition of ALDH1A1 decreases NF-κB/STAT3 signaling and increases pro-apoptosis signaling

Following ALDH1A1 knockdown, we also observed decreased levels of total NF-κB and STAT3, and phospho-NF-κB and -STAT3, compared with those in the control groups (Raji-wt and Raji-mock) (Fig. 4A and B, left panel and graph). Moreover, ALDH1A1 knockdown reduced BCL-2 levels and increased BAX, caspase-3 and −9 levels compared with those in the control groups (Raji-wt and Raji-mock) (Fig. 4A and B, right panel and graph). Inhibition of ALDH1A1 showed similar results (data not shown).

 Discussion

There is increasing evidence that ALDH1A1 expression is associated with poor prognosis in a variety of cancers. In a meta-analysis of 38 studies involving 6,057 patients, ALDH1A1 expression was significantly associated with lymph node metastasis, histological differentiation and clinical stage in lung and breast cancer (20). In the present study, ALDH1A1 was differentially expressed in peripheral blood samples from 112 NHL patients, compared with those in controls, and the median RQ level was 0.3263. Importantly, we further showed that high ALDH1A1 expression was associated with elevated levels of LDH, higher frequencies of >2 ECOG performance status and stage III/IV disease, higher IPI scores and a more invasive lymphoma category. Moreover, the overall survival time was significantly shorter in the high ALDH1A1 expression group than in the low ALDH1A1 expression group. Multivariate survival analysis further showed that the IPI score, lymphoma pathologic type and ALDH1A1 expression level were independent prognostic factors.

ALDH1A1 is now recognized as a CSC marker that is associated with malignant behavior and drug resistance in tumor cells. For instance, among Hodgkin's lymphoma cells, there is a subset of clonal CD27+/ALDH1A1high cells that are thought to be the initiating cells for HL (21). In addition, ALDH1 expression is higher in Epstein-Barr virus (EBV)-associated T/natural killer (NK)-cell lymphoproliferative disorder in children and young adults (TNKLPDC) than in extranodal nasal NK/T-cell lymphoma, and it is correlated with the biological characteristics of stem cells (22). There is also a subset of clonogenic ALDH+ cells in mantle cell lymphoma that are associated with multiple drug resistance (23). Our previous study demonstrated that ALDH1A1 mediates resistance of DLBCL cell lines Farage and Pfeiffer cells to CHOP treatment, which included cyclophosphamide, doxorubicin, vincristine and prednisone (15,17). Consistent with these previous studies, in the present study, we have similar conclusions. ALDH1A1 was upregulated in Raji cells, and inhibition of ALDH1A1 activity by DEAB increased the sensitivity of Raji cells to CHOP drugs. Furthermore, shRNA-mediated knockdown of ALDH1A1 decreased clonogenic ability, and increased apoptotic activity, in Raji cells. Next we should verify the conclusion in another non-aggressive NHL cell line to validate the effect of ALDH1A1.

NF-κB plays an important role in the development of B-cell NHL, and constitutive NF-κB activation is a major cause of drug resistance in relapse-refractory DLBCL patients. In a study using co-cultured engineered CD20-specific T cells with Raji cells, the T cells exerted antitumor activity against, and decreased the levels of p-STAT3 and BCL-2 in Raji cells potentially via inhibition of the NF-κB pathway (24). Moreover, invasive B-cell NHL is characterized by constitutive activation of NF-κB signaling; thus, targeting NF-κB is an attractive therapeutic strategy (25). NF-κB and STAT3 pathways interact with each other, and there are complex regulatory mechanisms between these signaling pathway networks. For instance, transglutaminase (TG2)/NF-κB and interleukin-6 (IL6)/STAT3 signaling cascades interact to promote autophagy and survival in mantle cell lymphoma, and blocking these pathways increases antitumor activity (26). Similarly, in DLBCL inhibition of an NF-κB/IL10/STAT3 autocrine loop is the main mechanism of drug-induced apoptosis (27). In the present study, we found that the levels of NF-κB/STAT3 pathway members and BCL-2 were decreased following ALDH1A1 knockdown, and concomitantly, the levels of the apoptosis-related proteins BAX, caspase-3 and −9 were increased.

In summary, the present study demonstrated that ALDH1A1 was associated with poor prognosis in NHL, and importantly, our data suggested that ALDH1A1 may be an independent prognostic indicator and a new molecular biomarker for diagnosis in NHL. Furthermore, we showed that inhibition of ALDH1A1 increased the sensitivity of NHL cells to chemotherapeutic drugs, further supporting the validity of ALDH1A1 as a potential therapeutic target in NHL treatment. Several inhibitors of ALDH1A1 could be used in the clinic (28); however, disulfiram (DSF), which is an oral drug that was formerly used in the treatment of chronic alcoholism, is particularly attractive since its antitumor effects have been confirmed in prostate, breast, lung and glioma (29–32). Recently, DSF has been applied in a phase II clinical study to treat prostate and lung cancer (ClinicalTrials.gov Identifier: NCT01118741, NCT00312819). Thus, these clinical studies by others provide evidence of the feasibility, and the present study provides evidence of the theoretical basis, for the promising use of ALDH1A1 inhibitors to treat NHL patients.

 Acknowledgements

The present study was supported by grants from the National Natural Science Foundation of China (no. 81570200).

 Competing interests

The authors declare that they have no competing interests.

 References ChiuBCWeisenburgerDDAn update of the epidemiology of non-Hodgkin's lymphomaClin Lymphoma4161168200310.3816/CLM.2003.n.02514715098 AdvaniACoiffierBCzuczmanMSDreylingMForanJGineEGisselbrechtCKettererNNastaSRohatinerASafety, pharmacokinetics, and preliminary clinical activity of inotuzumab ozogamicin, a novel immunoconjugate for the treatment of B-cell non-Hodgkin's lymphoma: Results of a phase I studyJ Clin Oncol2820852093201010.1200/JCO.2009.25.190020308665 MaxwellSAMousavi-FardSNon-Hodgkin's B-cell lymphoma: Advances in molecular strategies targeting drug resistanceExp Biol Med238971990201310.1177/1535370213498985 HuangXMengBIqbalJDingBBPerryAMCaoWSmithLMBiCJiangCGreinerTCActivation of the STAT3 signaling pathway is associated with poor survival in diffuse large B-cell lymphoma treated with R-CHOPJ Clin Oncol3145204528201310.1200/JCO.2012.45.6004242205633871513 BohersEMareschalSBouzelfenAMarchandVRuminyPMaingonnatCMénard ALEtancelinPBertrandPDuboisSTargetable activating mutations are very frequent in GCB and ABC diffuse large B-cell lymphomaGenes Chromosomes Cancer53144153201410.1002/gcc.2212624327543 DuongHQHwangJSKimHJKangHJSeongYSBaeIAldehyde dehydrogenase 1A1 confers intrinsic and acquired resistance to gemcitabine in human pancreatic adenocarcinoma MIA PaCa-2 cellsInt J Oncol41855861201210.3892/ijo.2012.1516227107323508685 SchäferATeufelJRingelFBettstetterMHoepnerIRasperMGemptJKoeritzerJSchmidt-GrafFMeyerBAldehyde dehydrogenase 1A1-a new mediator of resistance to temozolomide in glioblastomaNeuro Oncol1414521464201210.1093/neuonc/nos270231324083499020 ShienKToyookaSYamamotoHSohJJidaMThuKLHashidaSMakiYIchiharaEAsanoHAcquired resistance to EGFR inhibitors is associated with a manifestation of stem cell-like properties in cancer cellsCancer Res7330513061201310.1158/0008-5472.CAN-12-4136235423564506773 AlamgeerMGanjuVSzczepnyARussellPAProdanovicZKumarBWainerZBrownTSchneider-KolskyMConronMThe prognostic significance of aldehyde dehydrogenase 1A1 (ALDH1A1) and CD133 expression in early stage non-small cell lung cancerThorax6810951104201310.1136/thoraxjnl-2012-203021238781613841805 de BeçaFFCaetanoPGerhardRAlvarengaCAGomesMParedesJSchmittFCancer stem cells markers CD44, CD24 and ALDH1 in breast cancer special histological typesJ Clin Pathol66187191201310.1136/jclinpath-2012-20116923112116 ShenMBunaciuRPCongletonJJensenHASayamLGVarnerJDYenAInterferon regulatory factor-1 binds c-Cbl, enhances mitogen activated protein kinase signalingand promotes retinoic acid-induced differentiation of HL-60 human myelo-monoblastic leukemia cellsLeuk Lymphoma5223722379201110.3109/10428194.2011.603449217403033989140 IkedaJMamatSTianTWangYLuoWRahadianiNAozasaKMoriiEReactive oxygen species and aldehyde dehydrogenase activity in Hodgkin lymphoma cellsLab Invest92606614201210.1038/labinvest.2012.422349638 NeradugommaNKSubramaniamDTawfikOWGoffinVKumarTRJensenRAAnantSProlactin signaling enhances colon cancer stemness by modulating Notch signaling in a Jak2-STAT3/ERK mannerCarcinogenesis35795806201410.1093/carcin/bgt37924265293 LiuLSalnikovAVBauerNAleksandrowiczELabschSNwaeburuCMatternJGladkichJSchemmerPWernerJHerrITriptolide reverses hypoxia-induced epithelial-mesenchymal transition and stem-like features inpancreatic cancer by NF-κB downregulationInt J Cancer13424892503201410.1002/ijc.2858324615157 SongYHZhongMZGanPPYiPYTangYHLiuYPJiangJQLiLALDH1A1 mediates resistance of diffuse large B cell lymphoma to the CHOP regimenTumour Biol351180911817201410.1007/s13277-014-2335-925344211 FujitaSMoriiERahadianiNWadaNHoriYIkedaJIAozasaKSignificance of aldehyde dehydrogenase 1 expression in stromal cells of diffuse large B-cell lymphomaExp Ther Med2591594201110.3892/etm.2011.269229775453440706 JiangJLiuYTangYLiLZengRZengSZhongMALDH1A1 induces resistance to CHOP in diffuse large B-cell lymphoma through activation of the JAK2/STAT3 pathwayOnco Targets Ther953495360201610.2147/OTT.S107957276216505010157 WilsonWHTeruya-FeldsteinJFestTHarrisCSteinbergSMJaffeESRaffeldMRelationship of p53, bcl-2, and tumor proliferation to clinical drug resistance in non-Hodgkin's lymphomasBlood8960160919979002964 MaxwellSALiZJayeDBallardSFerrellJFuH14-3-3zeta mediates resistance of diffuse large B cell lymphoma to an anthracycline-based chemotherapeutic regimenJ Biol Chem2842237922389200910.1074/jbc.M109.022418195252242755960 ZhouYWangYJuXLanJZouHLiSQiYJiaWHuJLiangWClinicopathological significance of ALDH1A1 in lung, colorectal, and breast cancers: A meta-analysisBiomark Med9777790201510.2217/BMM.15.4926230297 JonesRJGockeCDKasamonYLMillerCBPerkinsBBarberJPValaMSGerberJMGellertLLSiednerMCirculating clonotypic B cells in classic Hodgkin lymphomaBlood11359205926200910.1182/blood-2008-11-189688191886632700327 NgSBOhshimaKSelvarajanVHuangGChooSNMiyoshiHShimizuNReghunathanRChuaHCYeohAEEpstein-Barr virus-associated T/natural killer-cell lymphoproliferative disorder in children and young adults has similar molecular signature to extranodal nasal natural killer/T-cell lymphoma but shows distinctive stem cell-like phenotypeLeuk Lymphoma5624082415201510.3109/10428194.2014.98309925382618 BrennanSKMeadeBWangQMerchantAAKowalskiJMatsuiWMantle cell lymphoma activation enhances bortezomib sensitivityBlood11641854191201010.1182/blood-2010-02-268375205708632993624 JiangLYuKDuJNiWHanYGaoSLiHWuJZhengYTanYInhibition of p38 MAPK activity in B-NHL Raji cells by treatment with engineered CD20-specific T cellsOncol Lett2753758201110.3892/ol.2011.308228482613406335 PhamLVTamayoATYoshimuraLCLoPTerryNReidPSFordRJA CD40 Signalosome anchored in lipid rafts leads to constitutive activation of NF-kappaB andautonomous cell growth in B cell lymphomasImmunity163750200210.1016/S1074-7613(01)00258-811825564 ZhangHChenZMirandaRNMedeirosLJMcCartyNTG2 and NF-κB signaling coordinates the survival of mantle cell lymphoma cells via IL6-mediated autophagyCancer Res7664106423201610.1158/0008-5472.CAN-16-0595274885295093039 EzellSAMayoMBihaniTTepsupornSWangSPassinoMGrosskurthSECollinsMParmentierJReimerCBythKFSynergistic induction of apoptosis by combination of BTK and dual mTORC1/2 inhibitors in diffuse large B cell lymphomaOncotarget549905001201410.18632/oncotarget.2071249708014148116 KoppakaVThompsonDCChenYEllermannMNicolaouKCJuvonenROPetersenDDeitrichRAHurleyTDVasiliouVAldehyde dehydrogenase inhibitors: A comprehensive review of the pharmacology, mechanism of action, substrate specificity, and clinical applicationPharmacol Rev64520539201210.1124/pr.111.005538225448653400832 LinJHaffnerMCZhangYLeeBHBrennenWNBrittonJKachhapSKShimJSLiuJONelsonWGDisulfiram is a DNA demethylating agent and inhibits prostate cancer cell growthProstate71333343201110.1002/pros.2124720809552 KimJYChoYOhELeeNAnHSungDChoTMSeoJHDisulfiram targets cancer stem-like properties and the HER2/Akt signaling pathway in HER2-positive breast cancerCancer Lett3793948201610.1016/j.canlet.2016.05.02627238567 LiuXWangLCuiWYuanXLinLCaoQWangNLiYGuoWZhangXTargeting ALDH1A1 by disulfiram/copper complex inhibits non-small cell lung cancer recurrence driven by ALDH-positive cancer stem cellsOncotarget758516585302016275422685295448 ParanjpeAZhangRAli-OsmanFBobustucGCSrivenugopalKSDisulfiram is a direct and potent inhibitor of human O6-methylguanine-DNA methyltransferase (MGMT) in brain tumor cells and mouse brain and markedly increases the alkylating DNA damageCarcinogenesis35692702201410.1093/carcin/bgt36624193513

ALDH1A1 mRNA levels were associated with prognosis in NHL patients. (A) ALDH1A1 mRNA levels in healthy controls (n=24), and NHL patients (n=112). The ALDH1A1 mRNA levels of NHL patients was significantly higher than healthy controls. (B) The median value of ALDH1A1 mRNA RQ was used to stratify patients into high and low ALDH1A1 expression groups. The cumulative survival of patients with low ALDH1A1 (<0.326) and high ALDH1A1 (>0.326) was determined by the Kaplan-Meier method.

(A and B) ALDH1A1 upregulated expression in Raji cells, shRNA silencing efficiency was confirmed by western blotting. The expression of ALDH1A1 in the Raji cells was upregulated 5.44-fold compared to B-cell. (A and B) The expression of ALDH1A1 in Raji-shRNA decreased 0.085-fold compared to Raji-mock after transfection with lentivirus particle shRNA. The IC50 values of CHOP were determined by CCK-8 assay. Cells were treated with increasing concentrations of CHOP for 48 h. The IC50 of Raji cells to CHOP regimen decreased from 685±25.62 to 412.56±38.58 ng/ml (P=0.013) using DEAB.

(A) After DEAB treatment, the numbers of colonies were significantly decreased whether adding CHOP or not (P<0.05). (B) ALDH1A1 shRNA Raji cells had a defect in clonogenic capacity compared with the mock-treated cells whether adding CHOP or not (P<0.05). (C) Caspase-3 activity assay showed that knockdown or inhibition of ALDH1A1 significantly increased apoptosis compared with the control or mock groups whether adding CHOP or not (P<0.05). (D) Caspase-9 activity assay showed knockdown or inhibition of ALDH1A1 significantly increased apoptosis compared with the control or mock groups whether adding CHOP or not (P<0.05). Annexin V and PI staining revealed that both knockdown of ALDH1A1 and drug treatments induced apoptosis. The upper left quadrant indicates late apoptosis, the lower left quadrant indicates early apoptosis. The apoptosis cells contained the early apoptosis and late apoptosis cells. The apoptosis rate of shRNA group was distinctly increased compared with wt or mock group (18.3±0.6 vs. 8.7±0.7/8.4±0.8%; P<0.01). (E) Moreover, shRNA group was significantly increased and the apoptosis rate after 400 ng/ml CHOP treatment for 24 h (50.1±1.1 vs. 18.3±0.6%; P<0.01).

Western blotting showed the total Stat3, NF-κB and phosphorylation-Stat3, phosphorylation-NF-κB (A), Bcl-2, Bax, caspase-3, caspase-9 (A) expressed in Raji cells. Knockdown or inhibition of ALDH1A1 had concordant trend. Both of them significantly decreased the total Stat3, NF-κB and phosphorylation-Stat3, phosphorylation-NF-κB, Bcl-2 levels and increased Bax, Caspase-3, Caspase-9 protein (B).

Correlation between ALDH1A1 expression and clinicopathological parameters in 112 NHL patients.

Low expression High expression
Clinical features N % N % χ2 P-value
Age (years) 0.801 0.502
  ≤60 45 52.33 41 47.67
  >60 11 42.31 15 57.69
Sex 1.885 0.239
  Female 24 58.54 17 41.46
  Male 32 45.07 39 54.93
B symptom 0.237 0.703
  Negative 32 51.61 30 48.39
  Positive 23 46.94 26 53.06
LDH 7.009 0.014
  Normal 36 62.07 22 37.93
  High 20 37.04 34 62.96
PS 7.467 0.011
  <2 42 51.22 28 48.78
  ≥2 14 33.33 28 66.67
Ann Arbor stage 14.756 0.000
  I–II 33 71.74 13 28.26
  III–IV 23 34.85 43 65.15
IPI score 14.583 0.000
  0–2 42 65.63 22 34.36
  3–5 14 29.17 34 70.84
Lymphoma category 12.341 0.001
  Indolent 30 71.43 12 28.57
  Progressive 26 37.14 44 62.86

χ2 test was used to compare the distribution of clinical features between ALDH1A1 low and high level expression groups. A P-value of <0.05 was considered significant. Low ALDH1A1 was <0.326, high ALDH1A1 was >0.326. ALDH1A1, aldehyde dehydrogenase 1A1; NHL, Non-Hodgkin's lymphoma; LDH, lactate dehydrogenase; PS, performance status; IPI, International Prognostic Index. Indolent lymphoma contained follicular lymphoma (n=18), marginal zone lymphoma (n=9), mucosa-associated lymphoid tissue type (n=11), unclassified small B cell lymphoma (n=4). Progressive lymphoma contained diffuse large B cell lymphoma (n=58), mantle cell lymphoma (n=9), Burkitt's lymphoma (n=3).

Multivariate analysis of factors contributing to overall survival in NHL patients.

Univariate analysis Multivariate analysis
Variables HR (95% CI) P-value HR (95% CI) P-value
Age, years (<60 vs. ≥60) 1.597 (0.756–3.376) 0.220 – –
Sex (female vs. male) 1.568 (0.725–3.391) 0.253 – –
B symptom (negative vs. positive) 1.352 (0.676–2.705) 0.393 – –
LDH (normal vs. high) 1.670 (0.823–3.387) 0.155 – –
PS (<2 vs. ≥2) 2.694 (1.334–5.440) 0.006 1.175 (0.520–2.657) 0.698
Ann Arbor stage (I–II vs. III–IV) 2.880 (1.29–6.428) 0.010 0.694 (0.230–2.096) 0.517
IPI score (0–2 vs. 3–5) 5.135 (2.340–11.266) 0.000 3.814 (1.262–11.523) 0.018
Lymphoma category 4.905 (1.719–13.993) 0.003 3.714 (1.254–11.005) 0.018
ALDH1A1 0.196 (0.089–0.431) 0.000 0.393 (0.160–0.968) 0.042

Univariate and multivariate analysis of prognostic factors in 112 NHL patients included in the survival analysis. Statistical analyses were performed by Cox proportional hazards regression. A P-value of <0.05 was considered significant. NHL, Non-Hodgkin's lymphoma; HR, hazard ratio; CI, confidence interval; LDH, lactate dehydrogenase; PS, performance status; IPI, International Prognostic Index.