Salvianolic acid A inhibits the growth of diffuse large B-cell lymphoma through MAPK pathways

•Salvianolic acid A reduced the viability of DLBCL cells in vitro.•Salvianolic inhibited tumor growth vivo.•Salvianolic regulated MAPK pathways cells. Treatment options are limited patients with diffuse large B-cell lymphoma (DLBCL). Salvianolic (SAA) is a water-soluble phenolic extracted from Salvia miltiorrhiza (Danshen) anti-tumor properties. The anti-leukemic activity SAA found our recent research prompted us to investigate therapeutic effect and mechanism action DLBCL. In work described here, we that by inducing cellular apoptosis, which was accompanied upregulation Bax cleavage PARP. Pre-incubation increased phosphorylation JNK, while it decreased p38 ERK Importantly, pharmacologic JNK inhibition partially mitigated anti-survival SAA, synergized SAA. Furthermore, suppressed xenograft mouse model vivo. Therefore, data suggest utility management Diffuse (DLBCL), an immense heterogeneous disease, can be categorized into two major molecular subtypes: germinal center B-cell-like (GCB) activated (ABC) [1Alizadeh AA Eisen MB Davis RE et al.Distinct types identified gene expression profiling.Nature. 2000; 403: 503-511Crossref PubMed Scopus (7778) Google Scholar]. This distinction represents lymphomas deriving different stages differentiation reflects fairly intracellular oncogenic for pathogenesis [2Lenz G Staudt LM. Aggressive lymphomas.N Engl J Med. 2010; 362: 1417-1429Crossref (463) Scholar, 3Roschewski M LM Wilson WH. lymphoma—treatment approaches era.Nat Rev Clin Oncol. 2014; 11: 12-23Crossref (275) 4Sehn LH Gascoyne RD. lymphoma: optimizing outcome context clinical biologic heterogeneity.Blood. 2015; 125: 22-32Crossref (355) Although combinatorial chemotherapy R-CHOP (rituximab cyclophosphamide, doxorubicin, vincristine, prednisone) makes majority curable [5Coiffier B Lepage E Briere al.CHOP plus rituximab compared CHOP alone elderly large-B-cell lymphoma.N 2002; 346: 235-242Crossref (4328) Scholar], subset exhibits severe side effects or relapses after initial treatment. Much worse, who fail have desperate prognosis. Thus, identifying more universal effective drugs high safety remains imminent objective. has been used treating cardiovascular diseases such as atherosclerosis myocardial infarction Asian countries many centuries, representing versatile safe traditional Chinese medicine. As one hydrophilic constituents this herb, salvianolic possesses diverse activities, including anti-thrombotic [6Huang ZS Zeng CL Zhu LJ Jiang L Li N Hu H. inhibits platelet activation arterial thrombosis via phosphoinositide 3-kinase.J Thromb Haemost. 8: 1383-1393Crossref (86) anti-inflammatory [7Wang K Yang Q Ma al.Protective against dextran sodium sulfate-induced acute colitis rats.Nutrients. 2018; 10: 791Crossref (36) Scholar,8Zhang HF Wang YL Gao C al.Salvianolic attenuates kidney injury inflammation inhibiting NF-kappaB signaling 5/6 nephrectomized rats.Acta Pharmacol Sin. 39: 1855-1864Crossref (34) anti-oxidative [9Zu Zhou T Che Zhang X Acid protects oxidative stress apoptosis induced intestinal ischemia-reperfusion through Nrf2/HO-1 pathways.Cell Physiol Biochem. 49: 2320-2332Crossref (31) cardioprotective [10Wang SB Tian S F HG XY Du GH. Cardioprotective on isoproterenol-induced rats.Eur Pharmacol. 2009; 615: 125-132Crossref (133) Recently, property growing. inhibitory proliferation multiple cell lines, HeLa, DU145, H1975, A549, reported [11Zhang Yu Y A, novel ETA receptor antagonist, shows vitro.Int Mol Sci. 2016; 17: 1244Crossref (12) also overcomes paclitaxel resistance human breast cancer [12Zheng Chen reverses migration invasion abilities inactivating transgelin 2.Cancer Biol Ther. 16: 1407-1414Crossref (40) Moreover, suppress oral squamous carcinoma [13Fang CY Wu CZ PN al.Antimetastatic potentials targeting MMP-2 c-Raf/MEK/ERK pathway.Environ Toxicol. 33: 545-554Crossref (21) Aside solid tumors, finding illustrates animal models, patient-derived primary leukemic [14Pei R Si Lu JX L. PI3K/Akt inhibitor, induces suppresses myeloid leukemia.Leuk Lymphoma. 59: 1959-1967Crossref (13) information implies potential value various cancers prompts further malignant lymphomas. present study indicates significantly vitro Mechanistic studies indicate involvement mitogen-activated protein kinase (MAPK) mediating growth-inhibitory purchased Shanghai Yi-Lin Biotech Company, Ltd (Shanghai, China). Its purity greater than 99% confirmed high-performance liquid chromatography. Primary antibodies PARP (No. 9532S), 5023S), Bcl-xL 2764S), Bcl-2 4223S), 9212S), phospho-p38 9216S), 4696S), phospho-ERK 4370S), 3708S), phospho-JNK 4668T), DR4 42533), DR5 8074), GAPDH 2118S) were Cell Signaling Technology (Beverly, MA). Human lines (OCI-Ly01, SUDHL-8, Farage, DB), Burkitt's line (Raji), mantle (Jeko-1) ATCC China) cultured RPMI-1640 medium (Hyclone, Logan, UT) supplemented 10% fetal bovine serum (FBS; AusgeneX, Molendinar, QLD, Australia), 100 U/mL penicillin, streptomycin, 2 mmol/L L-glutamine. Cells at 37°C 5% CO2 humidified incubator. Lymphoma seeded 96-well plates (10,000 μL per well) triplicates incubated phosphate-buffered saline (PBS) control. After culture indicated times, 10 MTS ([3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2- (4-sulfophenyl)-2H-tetrazolium, inner salt; Promega, Fitchburg, WI) added each well, incubation continued 4 hours. absorbance measured 492 nm using microplate reader (Molecular Devices, San Jose, CA). For assay, OCI-Ly01 SUDHL-8 six-well density × 105 well treated PBS 6 12 then washed stained fluorescein isothiocyanate (FITC)-conjugated Annexin V phycoerythrin (PE)-conjugated propidium iodide (PI) commercial kit (V13241, Invitrogen, Shanghai, according manufacturer's instructions. cycle analysis, fixed 70% cold ethanol. Then resuspended containing 1 mg/mL RNase (Sigma-Aldrich, St. Louis, MO), hour 37°C, adding 50 μg/mL (Sigma-Aldrich) 30 minutes 4°C dark. All samples acquired flow cytometry (CytoFLEX S, Beckman Coulter Life Sciences, Indianapolis, IN), analyzed FlowJo software (Tree Star, Carlos, lysed lysis buffer protease phosphatase inhibitors (Roche, Mannheim, Germany). total quantified BCA (TransGen Biotech, Beijing, instructions boiled 5 95°C. Equal amounts proteins separated dodecyl sulfate–polyacrylamide gel transferred polyvinylidene difluoride (PVDF) membrane. Membranes blocked milk hours respective overnight. corresponding secondary antibodies, goat anti-rabbit IR Dye 800CW anti-mouse 680 (LI-COR Biosciences, Lincoln, NE), membranes scanned densitometry Odyssey infrared imaging system Biosciences). use protocol approved Animal Care Committee Ningbo University School Medicine. Five-week-old male BALB/c nude mice (n = 15 line; Laboratory Center, subcutaneously injected right flank 106 PBS. tumor's maximal length width digital caliper, volume (V) calculated formula (length width2)/2. Once reached approximately mm3 (denoted day 0), distributed randomly three groups ≥4 group). (5 20 mg/kg) intraperitoneal injection. Tumors weights every 3 days, respectively. sacrificed 18 days administration. relative (RTV) RTV VX/V0 (VX represented recorded administration, V0 0). Data expressed means ± SD. Comparison between experimental performed Student's t test Prism package (Graph-Pad Software Inc., Diego, Differences mean values considered statistically significant p < 0.05. distinct subtypes responded differently. Aiming verify DLBCL, employed GCB-DLBCL OCI-Ly01, DB, ABC-DLBCL line, determined 24 hours, μmol/L but not 0.1 μmol/L, when PBS-treated control, evidenced decrease optical (OD) 0.597 0.045 0.236 0.069 (p 0.0001). suppressive became almost abolished concentration (Figure1A). To assess long-term impact treatment, 48 72 It observed exactly same 24-h incubation, suggesting most killed within they could no longer grow (Figure 1A). similar results obtained DB 1B–D). These capable killing regardless subtype. whether anti-growth specific Raji Jeko-1 used, these treatment (Supplementary Figure E1, online only, available www.exphem.org). We next examined had pro-apoptotic concentrations ranging tested V/PI dual staining cytometry. Compared controls, marked both types, reflected increases percentage V-positive 7.962 2.229 27.16 4.403 0.01) 10.2 4.854 30.45 8.203 0.05) However, did increase 2A). When time extended proportions apoptotic 70.66 24.52 64.84 16.72 cells, 90% underwent once 25 accordance GCB- exhibited equal susceptibility took Considering cytotoxicity might resulted induction arrest, doses distribution affected E2, suggested apoptosis. substantiate observations, evaluated level leukemia/lymphoma-2 (Bcl-2) family proteins, responsible regulating mitochondrial (intrinsic) pathway. comparison control led (a member family). influence levels (two anti-apoptotic members Cleavage PARP, steps involved under confirming 2B,C). true addition, (death receptors Apo2L/TRAIL) change E3, www.exphem.org), indicating non-mitochondrial (extrinsic) pathway contributed very little SAA-induced regulation cleavage. cascades essential benign lymphocytes [15Craxton Shu Graves JD Saklatvala Krebs EG Clark EA. required CD40-induced lymphocytes.J Immunol. 1998; 161: 3225-3236PubMed Scholar,16O'Reilly LA Kruse EA Puthalakath H al.MEK/ERK-mediated Bim ensure survival during mitogenic stimulation.J 183: 261-269Crossref (64) MAPKs death, lysates prepared, extracellular signal-regulated (ERK), (p38), c-Jun N-terminal (JNK) Western blot. illustrated 3, produced dose-dependent phosphorylation. Simultaneously, decreases lines. functionally confirm mediated signals, several selective blocking ERK, p38. SP600125, obvious dimethyl sulfoxide (DMSO)-treated made insensitive SP600125 only rescued possibility compensatory 4A,B). contrast, U0126 4C,D) SB203580 4E,F), respectively, enhanced addition all additive higher (50 μmol/L). aforementioned signals indeed SAA's vivo, murine established injecting BABL/c mice. Mice assigned receive mg/kg, intraperitoneally, daily) PBS, tumors palpable sizes. cell-implanted mice, grew rapidly, average ∼98-fold over expected, SAA-treated dramatically decelerated growth, administration mg/kg resulting 47-fold 29-fold volumes those prior respectively 5A). More importantly, weight loss among 5B). Accordingly, animals 5C,D). vivo responses consistent sensitivity treatment.Supplementary E1SAA cells.Show full captionRaji without (0.1-100 µmol/L). 24-h, 48-h, 72-h (OD 490 nm) assay. presented SD least independent experiments. ⁎P<0.05, #P<0.01, †P<0.001, ‡P<0.0001 versus points.View Large Image ViewerDownload Hi-res image Download (PPT)Supplementary E2SAA captionOCI-Ly01 (10, µmol/L) experiments.View E3SAA affect caption(A) Levels Representative blot images (B) Densitometric ratios shown (A).View (PPT) points. (A) (A). Since 1930s, 40 lipophilic Danshen, its biological functions [17Zhang P Ye Kim SH J. Tanshinones: sources, pharmacokinetics anti-cancer activities.Int 2012; 13: 13621-13666Crossref (161) well-defined activity, Danshen become attractive aspect recently. instance, tanshinone ⅡA induce autophagy osteosarcoma [18Huang ST Huang CC WL al.Tanshinone IIA intrinsic associated dysfunction.Sci Rep. 2017; 7: 40382Crossref Scholar,19Yen JH HS al.HGK-sestrin signaling-mediated contributes antitumor efficacy Tanshinone cells.Cell Death Dis. 9: 1003Crossref (30) Cryptotanshinone tongue single agent, synergizeed [20Wang HL Liu YD al.Cryptotanshinone sensitizes JAK/STAT3 pathway.Biomed Pharmacother. 95: 1388-1396Crossref (29) Isocryptotanshinone, analogue cryptotanshinone, lung, breast, gastric [21Guo Luo W al.Isocryptotanshinone, STAT3 pro-death A549 lung cells.J Drug Target. 24: 934-942Crossref 22Zhang Zhao X. Isocryptotanshinone MCF-7 Breast Cancer. 18: 112-118Crossref (15) 23Chen ZM MM Meng Ying SC Xu AM. 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Wong Magna Fund University. authors declare conflict interest.