Triptonide acts as a novel potent anti-lymphoma agent with low toxicity mainly through inhibition of proto-oncogene Lyn transcription and suppression of Lyn signal pathway

Accepted Manuscript Title: Triptonide acts as a novel potent anti-lymphoma agent with low toxicity mainly through inhibition of proto-oncogene Lyn transcription and suppression of Lyn signal pathway Authors: Ping Yang, Fulu Dong, Quansheng Zhou PII: DOI: Reference:

S0378-4274(17)30235-7 http://dx.doi.org/doi:10.1016/j.toxlet.2017.06.010 TOXLET 9797

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Toxicology Letters

Received date: Revised date: Accepted date:

1-3-2017 19-6-2017 27-6-2017

Please cite this article as: Yang, Ping, Dong, Fulu, Zhou, Quansheng, Triptonide acts as a novel potent anti-lymphoma agent with low toxicity mainly through inhibition of proto-oncogene Lyn transcription and suppression of Lyn signal pathway.Toxicology Letters http://dx.doi.org/10.1016/j.toxlet.2017.06.010 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

Triptonide acts as a novel potent anti-lymphoma agent with low toxicity mainly through inhibition of proto-oncogene Lyn transcription and suppression of Lyn signal pathway Running title: Triptonide is a novel potent anti-lymphoma agent. Ping Yang1,*, Fulu Dong2,*, Quansheng Zhou3,# 1

Department of Pathophysiology, Medical College, Nantong University, Nantong, Jiangsu, 226000, P.

R. China 2

Laboratory of Nuclear Receptors and Cancer Research, Center for Basic Medical Research, Medical

College, Nantong University, Nantong, Jiangsu, 226000, P. R. China 3

Cyrus Tang Hematology Center, Jiangsu Institute of Hematology, Soochow University; 2011

Collaborative Innovation Center of Hematology, Soochow University; Suzhou, Jiangsu 215123, P. R. China

#

Corresponding author: Quansheng Zhou

E-mail: [email protected] Phone：+86 (512) 65882116 Fax: +86 (512) 65880929 Mailing address: Cyrus Tang Hematology Center, Soochow University, 199 Ren Ai Road, Suzhou Industrial Park, Suzhou, 215123, China

*The authors contributed equally to this work.

Highlights for “Triptonide acts as a novel potent anti-lymphoma agent with low toxicity mainly through

inhibition of proto-oncogene Lyn transcription andsuppression of Lyn signal pathway”: 

Triptonide potently inhibits lymphoma cell proliferation..



Triptonide effectively suppresses lymphoma cell tumorigenicity.



Triptonide markedly inhibits proto-oncogene Lyn transcription.



4.Triptonide notably diminishesLyn downstream signal pathway.

1

Abstract Lyn is a proto-oncogene overexpressed and constitutively activated in lymphoma, and plays an important role in lymphoma initiation and malignant progression. Hence, the oncogenic Lyn has recently been targeted for novel anti-lymphoma drug discovery; however, the effective Lyn-targeted drug for lymphoma treatment with low toxicity is absent in the clinical setting. The goal of this study is to explore powerful and low toxic Lyn-targeted anti-lymphoma agent. Here we show that triptonide, a small molecule purified from the herb Tripterygium wilfordii Hook F, potently inhibits the proliferation of human B-lymphoma Raji and T-lymphoma Jurkat cells with IC50 of 5.7 nM and 4.8 nM, respectively. Strikingly, triptonide at a dose of 5 mg/kg/day almost completely inhibited the lymphoma growth in human lymphoma cells-xenografted mice without obvious side effects, particularly; the tumors in 6 mice among the 8 xenografted mice were completely eradicated.in vivo Cell biological studies showed that triptonide at the doses of 2.5-10 nM notably suppressed B-lymphoma cell colony-forming capability, and that triptonide at the dose of 20 nM promoted apoptosis through activation of PARP and caspase 3, but reduction of BCL2 protein levels in the lymphoma cells. Molecular studies revealed that triptonide markedly inhibited oncogenic Lyn transcription through suppressing the promoter activity of the gene, and that it remarkably reduced both total and phosphorylated Lyn proteins, and diminished Lyn downstream ERK and ATK signal pathways. Additionally, triptonide significantly enhanced p38 phosphorylation. Together, triptonide exerts potent anti-lymphoma effect with low toxicity mainly through inhibition of proto-oncogene Lyn transcription and suppression of Lyn downstream ERK and ATK signal pathways, providing an attractive drug candidate for development of novel anti-lymphoma therapeutics.

Key words: lymphoma; anti-cancer; Lyn; ERK, triptonide 1. Introduction Next-generation sequencing technology and gene expression profile analysis reveal that lymphoma is driven by multiple oncogenes derived from either poly-genetic mutation or deviant overexpression (Dunleavy et al., 2016; Rosenquist et al., 2016; Horn et al., 2017). These oncogenic genes aberrantly activates several key signal transduction pathways, such as PI3K-AKT (Bojarczuk et al., 2015), MAPK-ERK (Blachly and Baiocchi, 2014), JAK-STATs(Morimura et al., 2014; Deng et al., 2015), and NF-κB(Sasaki and Iwai, 2016). Among these oncogenic genes, Lyn (Lck/Yes-Related 2

Novel Protein Tyrosine Kinase) belongs to a proto-oncogene of the Src family kinase and is abnormally overexpressed and constitutively activated in lymphoma and leukemia (Prakash et al., 2005; Rovedo and Longnecker, 2008; Ingley, 2012). It has been well established that overexpression and sturdy activation of the oncogenic Lyn promote lymphoma cell proliferation, metastasis, and deficiency of apoptosis through escalation of several Lyn downstream signaling pathways, including ERK, AKT, and NF-κB (Prakash et al., 2005; Rovedo and Longnecker, 2008; Tauzin et al., 2008; Ke et al., 2009; Ingley, 2012; Leonard et al., 2016; Nguyen et al., 2016; Telford et al., 2016). In addition, clinical studies show that overexpression and robust activation of Lyn are closely associated with the poor prognosis of the patients suffered from lymphoma, leukemia, and various other malignant tumors (Martin et al., 2011; Wang et al., 2013). Accordingly, Lyn has recently been targeted for novel anti-lymphoma drug discovery and anti-lymphoma therapy (Kim et al., 2015). Several Lyn kinase activity inhibitors have been reported to exert anti-lymphoma, anti-leukemia and anti-cancer effects (Boukhiar et al., 2013; Kim et al., 2015; Huang et al., 2016; Leonard et al., 2016; Roskoski, 2016). Whereas, these Lyn-targeted drugs display either low efficacy or high toxicity in anti-lymphoma therapy; hence, effective Lyn-targeted drugs with low toxicity are highly desired. We recently explored novel anti-lymphoma agents from the traditional Chinese medicinal herbs and found that triptonide exerted potent anti-leukemia effect through the induction of leukemia cell senescence (Pan et al., 2016); whereas, the effect of triptonide on lymphoma has not been reported yet in the literatures. In the current investigation, we found that triptonide exerted extreme potent anti-lymphoma effect in vitro and in vivo mainly through suppression of oncogenic Lyn gene expression at the transcriptional level, inhibition of Lyn protein phosphorylation, and diminution of Lyn downstream ERK and AKT signal pathways, and an increase in p38 activation in human lymphoma cells, suggesting that triptonide is a new potent anti-lymphoma drug candidate for development of novel anti-lymphoma drug.

2. Materials and methods 2.1. Materials Triptonide (Purity ≥ 99%) was purchased from Chengdu Must Bio-technology Co., Ltd. (Chengdu, China). Alamar Blue® Assay Kit was from Invitrogen (Carlsbad, CA, USA). Annexin 3

V-FITC apoptosis detection kit was purchased from Biotech Co., Ltd (Urbana, USA). RNase-free DNase I was from Qiagen (Valencia, USA). Taq DNA Polymerase was from TaKaRa Biotechnology Co., Ltd (Dalian, China). RevertAid First Strand cDNA Synthesis Kit was from Fermentas Life Sciences (Maryland, USA). Methocult H4230 methylcellulose medium was from Stem Cell Technologies (Vancouver, Canada). The monoclonal antibody against β-actin was obtained from Sigma (St. Louis, MO USA). The antibodies against caspase 3, cleaved caspase 3, PARP, total Akt, phospho-Akt (Thr308), total p38, phospho-p38 (Thr180/Tyr182), total Erk1/2, phospho-Erk1/2 (Thr202/Tyr204), total NF-κB, and phospho-NF-κB (Ser536), were from Cell Signaling Technology (Beverly, MA, USA). Lyn antibody was from Santa Cruz biotechnology (SantaCruz, CA, USA). Human lymphoma cell lines Raji and Jurkat were obtained from ATCC (Manassas, USA). PGL4.17 vector was from Promega (Madison, Wisconsin, USA). 2.2. Cell culture Human lymphoma cell lines Raji and Jurkat were cultured in RPMI-1640 medium supplemented with 10% heat-inactivated bovine serum, 100 U/ml penicillin G and 100 µg/ml streptomycin under a humidified atmosphere of 5% CO2 at 37 °C as we previously described (Yang et al., 2015)Yang, et al., 2015. 2.3. Cell proliferation assay Cell proliferation was determined by Alamar Blue assay as we reported previously(Feng et al., 2012; Cao et al., 2013; Shang et al., 2014). In brief, 200 µl of lymphoma Raji and Jurkat cells were cultured in T25 flask with either DMSO as control or triptonide at the concentrations of 0-80 nM. After incubation for either 3 days or 6 days, 10µl of Alamar Blue solution was added to each well. The plates were incubated for another 2 h at 37 °C℃ and 5% CO2, and absorbance at 560 nm and 590 nm was measured using a SpectraMax M5 multi-detection reader. 2.4. Colony forming assay Colony forming assay was performed as we described before (Yang et al., 2015; Pan et al., 2017) In brief, lymphoma Raji cells were incubated with triptonide at concentrations of 0-10 nM for 6 days, then washed in fresh medium, and 1

× x 103 living cells were mixed with Methocult H4230

methylcellulose medium and plated in 30-mm plastic dishes. After incubation for 14 days, colonies were counted and imaged using a dissecting microscope (SZX16, OLYMPUS). 2.5. Mouse tumor xenograft 4

All mice used in this study were maintained in a laminar airflow cabinet under specific pathogen-free conditions in a 12-h light-dark cycle, and animal care and experiments followed approved animal protocols of Soochow University Animal Care and Use Committee (Yang et al., 2015; Pan et al., 2017). Eight-week-old female NOD/SCID mice (18-22g) were randomly divided into two groups, 8 mice each group. Each mouse was subcutaneously injected with 3 x 107 Raji cells in 200 µL saline at the back, and then intraperitoneally injected daily with either triptonide at the dose of 5 mg/kg/d or saline as control. The body weight of each mouse was recorded and the tumor volume was monitored using digital caliper every other day. Tumor growth was calculated according to the formula: tumor volume = 0.55 x length x width2. After treatment with triptonide for 35 days, blood samples were collected from the eye veins of the mice and put into the blood collection tubes containing sodium citrate solution, measured by Automated Hematology Analyzer KX-21N (Sysmex Corporation). Then the mice were sacrificed, solid tumors were excised, weighed, and imaged, and the heart, liver, spleen, lung, and kidney of each mouse were also excised, weighed. The coefficient of organs (organ index) was analyzed as follows: organ index = weight of organ/weight of body weight x 1000. 2.6. Cell apoptosis assay Apoptotic cells were assayed using Annexin V-PI staining kit as previously described(Feng et al., 2012; Shang et al., 2014). Briefly, Raji cells were exposed to triptonide at concentrations of 0-20 nM for 3 days. The cells were harvested, washed, and re-suspended with phosphate buffered saline (PBS). Apoptotic or necrotic cells were stained with fluorescein isothiocyanate (FITC)-labeled Annexin V and PI, and analyzed by a flow cytometry (Becton Dickinson FACSCalibur, BD Biosciences, New Jersey, U.S.A.). 2.7. Western blotting Raji cells were lyzed by M-PER Mammalian Protein Extraction Kits. For analysis of the signaling protein phosphorylation, Raji cells were lyzed by the lysis buffer with 1% SDS, 10 mM NaV, 10 mM NaF and 10mM DTT. Cell lysates were loaded into each lane and resolved by sodium dodecyl sulfate polyacrylamide gel electrophoresis with Tris-glycine running buffer and transferred to nitrocellulose membranes. Membranes were blocked with 5% nonfat milk and incubated overnight at 4 °C with the primary antibodies against a protein, followed by incubation with HRP-coupled secondary antibody for 1 h at room temperature. Blots were visualized using enhanced 5

chemiluminescence (ECL) detection reagents and exposed to x-ray film. The blots were stripped and re-probed with the HRP-coupled anti-β-actin antibody. 2.8. Semi-quantitative RT-PCR and quantitative real-time PCR Total RNA was extracted from triptonide-treated Raji cells. cDNA was generated by reverse transcription (RT) using RevertAid First Strand cDNA Synthesis Kits and oligo (dT) primers in 20 µL reactions containing 5 µg total RNA pretreated with RNase-free DNase I. Semi-quantitative RT-PCR (RT-PCR) was performed with 25 µL reactions containing 1 µL of diluted cDNA, and 1 µL of TaKaRa Taq DNA Polymerase. The RT-PCR reaction consisted of an initial denaturation at 94°C for 4 min, followed by 25-35 cycles of 94°C for 30 s, 58–68°C for 30 s, and 72°C for 1 min. Quantitative real-time PCR (QT-PCR) was carried out with 25 µL reactions containing 1 µL of diluted cDNA, and 1 µL of SYBR Green Mix. The reaction consisted of an initial denaturation at 95°C for 4 min, followed by 40 cycles of 94°C for 15 s, 60°C for 30 s, and 72°C for 30 s. RT-PCR products were analyzed on 1.5% agarose gels and QT-PCR was analyzed by real-time PCR machine (7500, ABI). Primers used in the PCR were listed in the Supplemental Table 1. 2.9. Luciferase assay Lyn gene promoter DNA (-520bp to +453bp) was first amplified by RT-PCR with the template of genomic DNA, then cloned to luciferase reporter system PGL4.17 vector. The Raji cells were transfected the Lyn gene promoter DNA-PGL4.17 by Lipofectamine 2000 Reagent kit (Invitrogen, Carlsbad, USA), and the stably transfected cells were selected by G418. After treatment of the stably

transfected cells by triptonide at the doses of 0-10 nM, the luciferase activity in cell lysates was measured using Dual-luciferase Reporter assay kit (Promega, Madison, USA).

2.10. DNA microarray DNA microarray was performed as we previously described (Shang et al., 2014). In short, Raji cells were divided into 6 groups, the cells in 3 groups were treated with triptonide at the dose of 6 nM in the complete medium for 6 days, and the other 3 groups were incubated the complete medium as control. The cells were collected and lyzed by the Trizol solution. DNA microarray was performed by Shang Hai Biotechnology Corporation using Affymetrix Human U133 Plus 2.0 Array. with average linkage and uncentered correlation as the similarity metrics using Cluster 3.0.Differentially expressed genes were identified using the significance analysis of microarrays (SAM) program. The genes with expression levels either genes with q-Value ≤ 0.05, fold Change>≥ 32 or <≤ 0.335 fold with P < 6

0.05 were considered to be significantly differentially expressed.

Differentially expressed gene Lyn

was further confirmed by both RT-PCR and Western blotting as mentioned above. 2.11. Statistic analysis The data shown in this study represented the mean ± S.D. Differences between the groups were assessed by one-way ANOVA using SPSS 16.0 software. The significance of differences was indicated as *P < 0.05 and **P < 0.01.

3. Results 3.1. Triptonide potently inhibits lymphoma cell tumorigenic capability with low toxicity. We scanned potent anti-lymphoma component from the traditional Chinese medicinal herbs and found that triptonide, a small molecule purified from the herb Tripterygium wilfordii Hook F with a molecular weight of 358.39 (Figure 1A), had very strong growth-inhibitory effect on multiple lymphoma cell lines. In light of the anti-lymphoma effect of triptonide has not been reported yet, we first evaluated the anti-lymphoma effect of triptonide in vitro using human B-lymphoma Raji and T-lymphoma Jurkat cell lines. Cell proliferation assay using Alamar Blue method showed that these two lymphoma cell lines were very sensitive to triptonide in a dose-dependent manner, with the IC50 of 11.4 nM at 3 days and 5.7 nM at 6 days in B-lymphoma Raji cells (Figure 1B), and 11.1 nM at 3 days and 4.8 nM at 6 days in T-lymphoma Jurkat cells (Figure 1C), respectively. Next, we investigated whether the triptonide-mediated anti-lymphoma cell growth effect is through induction of cell apoptosis. Annexin V-PI staining and follow cytometry analysis indicated that triptonide did not significantly induce apoptosis at the effective tumor growth-inhibitory doses of 2.5-10 nM; whereas, it moderately induced lymphoma cell apoptosis at the concentration of 20 nM (Figure 1D and 1E). Consistently, Western blot displayed that pro-apoptotic proteins PARP and caspase 3 in lymphoma cells were not vigorously activated by triptonide at the doses of 2.5-10 nM, however, both PARP and caspase 3 were significantly activated by triptonide at the dose of 20 nM; additionally, anti-apoptotic BCL2 levels were significantly reduced by triptonide (Figure 1F). These data indicate that induction of apoptosis plays a minor role in triptonide-exerted anti-lymphoma effect at the low doses of 10 nM and less, but triptonide at the dose of 20 nM significantly induces a moderate apoptosis, implying that other unknown mechanism other than apoptosis may be responsible for the anti-lymphoma effect of the low dose of triptonide. 7

To unravel the mystery of low dose of triptonide-exerted anti-lymphoma effect, we examined lymphoma cell morphological alterations and noted that the morphology of Raji and Jurkat cells was obviously changed after treatment of the cells with triptonide at the low doses of 2.5-10 nM, especially, the cell size and cell nucleus were remarkably enlarged in an irregular form (Figure 2A). More interestingly, the lymphoma cell colony formation assay displayed that the colony numbers were significantly reduced by triptonide at the doses of 2.5–10 nM compared to the control without triptonide; notably, triptonide at the dose of 10 nM almost completely inhibited the colony-forming capability of Raji lymphoma cells (Figure 2B and 2C). Following, we evaluated anti-lymphoma effect of triptonide in vivo using human lymphoma cell xenograft model. After the human lymphoma cell-xenografted NOD/SCID mice were daily treated with triptonide at the dose of 5 mg/kg for 34 days, the tumors in 6 mice among the 8 xenografted mice were completely eradicated, while the tumors in the left two mice were markedly reduced; on contrast, the tumor grew up rapidly in the saline control mice (Figure 3A and 3B). After triptonide treatment for 34 days, the average tumor weights of the triptonide-treated and saline control mice were 50.8±10.5 mg and 1088.75±68.5mg, respectively (Figure 3C). Of note, after the mice were treated with triptonide for 34 days, the organ indexes of the heart, liver, lung, and kidney of the mice did not significantly change except for a moderate increase in the spleen (Figure 3D); additionally, the red blood cell (RBC) count, hemoglobin (HGB) level were not significantly changed (Table 1); whereas, platelet (PLT) count was moderately increased in the blood. Interestingly, while blood cell (WBC) count was 36% higher in the triptonide-treated mice as compared with the control mice, although it did not reach the statistical significance (P > 0.05), suggesting that triptonide may fairly elevate WBC levels in vivo. Collectively, these data indicate that triptonide potently inhibits lymphoma cell growth and tumorigenic capability, and exerts a strong anti-lymphoma effect in mice without obvious toxicity at the dose tested. 3.2. Triptonide inhibits oncogenic Lyn expression and diminishes Lyn downstream signal pathways in lymphoma cells To elucidate the mechanisms of triptonide-mediated anti-lymphoma effect, we performed DNA microarray to analyze the differential gene expression profile between the triptonide-treated and control human B-lymphoma Raji cells. The results showed that 418 genes were up-regulated, while 667 genes were down-regulated for 3 fold and above after the cells were treated with 6 nM triptonide 8

(data not shown), and among the triptonide down-regulated genes, the expression of the pro-oncogene Lyn was reduced 4.5 fold. In light of that the oncogenic Lyn plays an important role in the development of lymphoma (Prakash et al., 2005; Rovedo and Longnecker, 2008; Tauzin et al., 2008; Ke et al., 2009; Martin et al., 2011; Ingley, 2012; Wang et al., 2013; Leonard et al., 2016; Nguyen et al., 2016; Telford et al., 2016), we focused on investigation of the down-regulation of Lyn by triptonide in the current study. RT-PCR indicated that triptonide at the doses of 5 and 10 nM significantly diminished Lyn mRNA levels in the lymphoma cells (Figure 4A and 4B). Quantitative real-time PCR showed that the Lyn mRNA levels in the lymphoma cells treated with 6 nM triptonide were decreased for 5.7 fold as compared with the control with triptonide (Figure 4C). Similarly, Lyn protein levels were also strikingly decreased (Figure 4D and 4E). In addition, the phosphorylation of Lyn was also significantly reduced by triptonide (Figure 4D and 4F). Next, we used luciferase reporter system to examine whether triptonide-reduced Lyn expression was through transcriptional suppression. The luciferase assay indicated that the Lyn gene promoter activity in lymphoma cells was reduced approximate 5 fold after 10 nM triptonide treatment (Figure 4G and 4H), suggesting that triptonide-mediated down-regulation of Lyn expression is mainly through reduction of Lyn gene transcription. In view of that Lyn is a proto-oncogene and functions as a key signaling protein underneath the inner leave of cell membrane, and that activation of Lyn escalates several downstream signal pathways, such as ERK, AKT, and NF-κB to trigger carcinogenesis, we investigated whether the down-regulation of Lyn expression and reduction of Lyn phosphorylation results in diminution of Lyn downstream signal transduction. Western blotting showed that the phosphorylated ERK (pERK) protein was significantly decreased in a triptonide dose-dependent manner, while the total ERK protein was not obviously affected (Figure 5A and 5B), suggesting that triptonide inhibits both Lyn expression and activation; additionally, total and phosphorylated AKT were significantly diminished by 10 nM triptonide (Figure 5A and 5C). On contrast, phosphorylated p38 protein levels were significantly increased, but the total p38 levels were not clearly changed in lymphoma cells after triptonide treatment (Figure 5A and 5C). Several other key signaling proteins including CREB, NF-κB, and STAT3 were not obviously affected (Figure 5A). These data suggest that triptonide at the low doses of 5 and 10 nM mainly selectively inhibits Lyn down-stream ERK and AKT signaling pathways, and activates p38 signaling. 9

Taken together, these data indicate that triptonide exerts potent anti-lymphoma effect through repression of oncogenic Lyn expression, inhibition of Lyn phosphorylation, diminution Lyn downstream ERK and AKT signal pathways, and enhancement of p38 phosphorylation in lymphoma cells (Figure 6).

4. Discussion It has been reported that triptonide displays insecticidal and immune-moderation effects (Luo et al., 2004; Peng et al., 2008; He et al., 2015). More recently, Chinison J et al reported that triptonide induced apoptosis of cancer cell lines in vitro(Chinison et al., 2016), and we uncovered that triptonide strongly induced complete senescence of leukemia cells and almost completely suppressed leukemia cell tumorigenicity in the xenografted mice without obvious complications (Caunt et al., 2015). In the current investigation, we found that triptonide exerted potent anti-lymphoma effect in vitro and in vivo with low toxicity. This is the first report to address the anti-lymphoma effect of triptonide. As a new anti-lymphoma agent, triptonide has several advantages over other Lyn inhibitors and Lyn-based lymphoma therapeutics (Boukhiar et al., 2013; Kim et al., 2015; Huang et al., 2016; Leonard et al., 2016; Roskoski, 2016). First of all, triptonide is a novel potent anti-lymphoma agent with IC50 around 5 nM and has high efficacy in the inhibition of lymphoma growth in the xenografted mice, much stronger than many other anti-lymphoma drugs. Lyn protein is located at the inner leaf of cell membrane and belongs to upstream of multiple signal pathways such as ERK and AKT signaling; hence, suppression of Lyn by triptonide works effectively and efficiently in anti-lymphoma. Secondly, triptonide exerts anti-lymphoma mainly through inhibition of oncogenic gene Lyn expression, distinct from all of the Lyn protein kinase inhibitors which usually suppress several Src family and other protein kinases, resulting in side effects. The third, triptonide at the dose as low as 10 nM exerts strong anti-lymphoma effect mainly by inhibition of oncogenic proto-oncogene Lyn transcription and activation, diminution of Lyn downstream signal pathways, leading to potent anti-tumorigenic effect, but not strongly induces cell apoptosis and causes obvious side effects in mice at the effective anti-lymphoma dosage, consistent with the low toxicity of triptonide in the mice reported by Xu L et al (Xu et al., 2013) and Pan Y et al (Pan et al., 2017). Of note, both the blood WBC and platelet numbers are moderately increased in the mice after triptonide treatment; apparently, triptonide does not brings about pancytopenia, a conmen side effect 10

caused by many currently used cytotoxic anti-lymphoma and anti-cancer drugs which induce bone marrow cell apoptosis and reduce blood WBC and platelet counts, resulting in inflammation or bleeding complications in the cancer patients. Thus, high anti-lymphoma efficacy with low toxicity lets triptonide to stand as an attractive anti-lymphoma drug candidate, warranting for further development of novel anti-lymphoma drug. Although we reveal that triptonide strongly inhibits Lyn gene transcription which is distinct from current Lyn protein kinase-targeted agents and drugs (Boukhiar et al., 2013; Kim et al., 2015; Huang et al., 2016; Leonard et al., 2016; Roskoski, 2016), the molecular mechanism of triptonide-mediated inhibition of Lyn gene transcription in lymphoma cells are unclear; hence, this is the limitation of the current study. By the way, in parallel to study the anti-lymphoma effect of triptonide, we have recently made all-out to identify the receptor of triptonide in malignant tumors and found triptonide binds to several candidate proteins (data not shown); whereas, the receptor of triptonide in lymphoma cells remains to be deeply investigated. Further study of the interaction between triptonide and its receptor in lymphoma cells will not only get new insight into the triptonide-mediated anti-lymphoma, but also provide new target for novel anti-lymphoma and anti-cancer drug discovery.

Disclosure of Potential Conflicts of Interest No potential conflicts of interest were disclosed.

Acknowledgments This study was supported by grants from National Natural Science Foundation of China (Grants No. 81372376, No. 81572257, No. 81071306, No.81300553, and No.81503304), a project funded by the priority academic program development of Jiangsu Higher Education Institutions (PAPD), 2011 Collaborative Innovation Center of Hematology, Soochow University, and Jiangsu Key Laboratory of Preventive and Translational Medicine for Geriatric Diseases, Natural Science Funds of Jiangsu Colleges and Universities (No.16KJB310015), Suzhou City Scientific Research Funds (No. SYS201418), Natural Science Funds of Soochow University (No. SDY2014A22).

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Reinart, N., Hallek, M., 2016. LYN Kinase in the Tumor Microenvironment Is Essential for the Progression of Chronic Lymphocytic Leukemia. Cancer cell 30, 610-622. Pan, Y., Meng, M., Zheng, N., Cao, Z., Yang, P., Xi, X., Zhou, Q., 2017. Targeting of multiple senescence-promoting genes and signaling pathways by triptonide induces complete senescence of acute myeloid leukemia cells. Biochemical pharmacology 126, 34-50. Peng, A., Li, R., Hu, J., Chen, L., Zhao, X., Luo, H., Ye, H., Yuan, Y., Wei, Y., 2008. Flow rate gradient high-speed counter-current chromatography separation of five diterpenoids from Triperygium wilfordii and scale-up. Journal of chromatography. A 1200, 129-135. Prakash, O., Swamy, O.R., Peng, X., Tang, Z.Y., Li, L., Larson, J.E., Cohen, J.C., Gill, J., Farr, G., Wang, S., Samaniego, F., 2005. Activation of Src kinase Lyn by the Kaposi sarcoma-associated herpesvirus K1 protein: implications for lymphomagenesis. Blood 105, 3987-3994. Rosenquist, R., Rosenwald, A., Du, M.Q., Gaidano, G., Groenen, P., Wotherspoon, A., Ghia, P., Gaulard, P., Campo, E., Stamatopoulos, K., European Research Initiative on, C.L.L., the European Association for, H., 2016. Clinical impact of recurrently mutated genes on lymphoma diagnostics: state-of-the-art and beyond. Haematologica 101, 1002-1009. Roskoski, R., Jr., 2016. Ibrutinib inhibition of Bruton protein-tyrosine kinase (BTK) in the treatment of B cell neoplasms. Pharmacological research 113, 395-408. Rovedo, M., Longnecker, R., 2008. Epstein-Barr virus latent membrane protein 2A preferentially signals through the Src family kinase Lyn. Journal of virology 82, 8520-8528. Sasaki, Y., Iwai, K., 2016. Roles of the NF-kappaB Pathway in B-Lymphocyte Biology. Current topics in microbiology and immunology 393, 177-209. Shang, B., Gao, A., Pan, Y., Zhang, G., Tu, J., Zhou, Y., Yang, P., Cao, Z., Wei, Q., Ding, Y., Zhang, J., Zhao, Y., Zhou, Q., 2014. CT45A1 acts as a new proto-oncogene to trigger tumorigenesis and cancer metastasis. Cell death & disease 5, e1285. Tauzin, S., Ding, H., Khatib, K., Ahmad, I., Burdevet, D., van Echten-Deckert, G., Lindquist, J.A., Schraven, B., Din, N.U., Borisch, B., Hoessli, D.C., 2008. Oncogenic association of the Cbp/PAG adaptor protein with the Lyn tyrosine kinase in human B-NHL rafts. Blood 111, 2310-2320. Telford, N., Alexander, S., McGinn, O.J., Williams, M., Wood, K.M., Bloor, A., Saha, V., 2016. Myeloproliferative neoplasm with eosinophilia and T-lymphoblastic lymphoma with ETV6-LYN gene fusion. Blood cancer journal 6, e412. 14

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15

Figure legends

Figure 1 Triptonide potently inhibits the proliferation of human lymphoma cells and induces apoptosis. The chemical structure of triptonide is shown in Figure 1A. Human B-lymphoma Raji (Figure 1B) and T-lymphoma Jurkat cells (Figure 1C) were incubated with triptonide at the concentrations of 0-80 nM for 3 days (black line) and 6 days (red line), respectively, and the IC50 (half maximal inhibitory concentration) was analyzed. The apoptosis of Raji cells was stained with FITC-Annexin V and PI, analyzed by flow cytometry. The apoptotic (right-up and right-low quadrants) and necrotic Raji cells (left-up quadrant), and surviving Raji cells (left-low quadrant) were shown in the quadrants each figure (Figure 1D), and statistically analyzed (Figure 1E). Additionally, the levels of key apoptosis-regulatory proteins PARP, caspase 3, and BCL2 were detected using Western blotting (Figure 1F). The data represent three independent experimental repeats. *P < 0.05 and **P < 0.01.

Figure 2. Triptonide causes cell morphological changes and markedly diminishes lymphoma cell colony-forming capability. The morphology of human B-lymphoma Raji (Figure 2A) and T-lymphoma Jurkat cells

(Figure 2B) were imaged under a microscope (1000 x), the enlarged cells and cell nuclei were indicated by red arrows. The effect of triptonide on tumorigenic capability of the lymphoma cells was measured by the colony-forming assay, and the colonies in the five random-selected fields were imaged, counted (Figure 2C), and statistically analyzed (Figure 2D). The data are representative of three repeats. *P < 0.05 and **P < 0.01.

Figure 3. Triptonide almost completely suppresses human lymphoma cell tumorigenicity and tumor growth in the xenografted mice. NOD/SCID mice were subcutaneously injected with B-lymphoma Raji cells, followed by daily injection of triptonide at a dosage of 5 mg/kg or saline as control. The tumor volume was measured every other day (Figure 3A). After treatment of the mice with triptonide for 34 days, the tumors in the mice were excised, imaged (Figure 3B), and weighted (Figure 3C). After the mice were dissected, the organs of the mice were weighed, and the organ index was calculated as follows: Organ index = 16

weight of organ/weight of mouse body weight x 1000 (Figure 3D). The data show mean ± SD, *P <

0.05 and **P < 0.01.

Figure 4. Triptonide inhibits Lyn gene expression in lymphoma Raji cells. The Raji cells were treated with triptonide at the doses of 0-10 nM for 72 hours, The Lyn gene expression in the cells was detected by RT-PCR (Figure 4A), and the bands were scanned and analyzed using Quantity One software (Figure 4B). The Lyn expression in Raji cells was also determined by quantitative real time-PCR (QT-PCR) (Figure 4C). Additionally, the phosphorylated Lyn and total Lyn proteins in the cells was detected by Western blotting (Figure 4D), followed by statistical analysis (Figure 4E and 4F). The putative transcription factor binding sites in the major promoter region of Lyn gene (-520bp to +453bp) were analyzed by bioinformatics, and indicated in Figure 4G. The effect of triptonide on Lyn gene promoter activity was measured by luciferase assay, and the data were statistically analyzed (Figure 4H). The data are representative of three repeats. *P < 0.05 and **P < 0.01.

Figure 5.

Triptonide diminishes Lyn downstream ERK and AKT signaling pathways and

promotes p38 phosphorylation in lymphoma cells. After the Raji cells were treated with triptonide at the doses of 0-10 nM for 72 hours，the total and phosphorylated proteins of ERK1/2, AKT, p38, NF-κB, and STAT3 were detected by Western blotting (Figure 5A). The integrated optical band density of p-ERK1/2 (Figure 5B), p-AKT (Figure 5C), and p-p38 (Figure 5D) was analyzed by scanning and Quantity One software. The data are representative of three repeats. *P < 0.05 and **P < 0.01.

Figure 6. Mechanistic summary of triptonide-mediated anti-lymphoma Triptonide binds to intracellular unknown receptor (? marker), causes transcriptional inhibition of Lyn and AKT genes, and diminishes Lyn protein phosphorylation, resulting in suppression of Lyn downstream ERK and AKT signaling pathways; on the other hand, triptonide enhances p38 phosphorylation. Consequently, triptonide potently inhibits lymphoma cell growth, suppresses lymphoma cell tumorigenicity, and induces tumor cell apoptosis.

17

Figure 1 B. Raji cells 100% 100%

Inhibitory rate(%)

A

80%80%

Inhibitory rate(%)

3 days

60%60%

IC50=11.4nM IC50=5.7nM

40%40% 20%20% 0%0%

Triptonide(MW=358.39) C. Jurkat cells 120.00% 100%

6 days

00

20 20

40 60 40 60 Triptonide (nM)

80 80

6 days

100.00% 80% 80.00%

3 days IC50=11.1nM IC50=4.8nM

60%

60.00%

40%

40.00%

20% 20.00% 0.00% 0% 0 0

4.61% 0.90%

103

40 60 40 60 Triptonide (nM)

80 80

20 nM

10 nM 11.16% 0.60%

5 nM 4.38% 0.15%

33.78% 1.31%

102 101 87.34% 0.81% 95.21% 0.26% 94.14% 0.35% 63.83% 1.08% 100 0 0 2 3 4 0 1 0 2 3 4 1 2 3 4 1 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 101 102 103 104 PI E

Apoptosis cells (%)

Annexin V

D. Triptonide 0 nM 104

2020

F 100.00% 100

PARP

80.00% 80

Caspase3

60.00% 60

40 40.00%

Cleaved Caspase3 β-actin

**

20 20.00% 00.00%

00

55 10 20 10 20 Triptonide (nM) 18

Triptonide: (nM)

0

5

10

20

Figure 2 A. Raji cells Triptonide 0 nM

2.5 nM

5 nM

10 nM

Triptonide 0 nM

2.5 nM

5 nM

10 nM

Triptonide 0 nM

2.5 nM

5 nM

10 nM

v

B. Jurkat cells

C

D

Colony numbers

250 250 200 200

*

150 150 100 100

**

5050 0

** 0

01

2.5 2 35 Triptonide (nM)

104

19

Figure 3 B 2

Tumor weight (g)

2500 2500 0 mg/kg 5 mg/kg

2000 2000 1500 1500 1000 1000 500500 00

C

1.5 1 0.5 0

0 0

55

1010

15 20 15 20 Days

25 25

30 30

3535

** 0 5 Triptonide（mg/kg）

Triptonide (mg/kg) 0 5

D 0.1

1.0

Orgen index unit

Tumor volume (mm3)

A

0.08 0.8

0mg/kg

0.06 0.6

5mg/kg

0.04 0.4

0.2 0.02 00

** Heart

心脏系数

Liver

肝脏系数

Spleen

脾脏系数

20

Lung

肺脏系数

Kidney 肾脏系数

Figure 4 B 3.0 3 2.5 2.5 2.0 2 1.5 1.5 1.0 1 0.5 0.5 00

RT-PCR

Lyn/ β-actin

A Lyn β-actin Triptonide: (nM)

0

5

10

* **

1

QT-PCR

p-Lyn

1.21.2

T-Lyn

0.80.8 0.40.4

β-actin

**

00

Triptonide: (nM)

01 62 Triptonide (nM)

E

F 5 5.0 4 4.0

3.0 3 2.0 2

*

1.0 1

**

0

0

0

1

5 10 3 Triptonide (nM)

3 3.0 2.5 2.5 2.0 2 1.5 1.5 1.0 1 0.5 0.5 00 1

H Lymphokine element

C

MYB

MYB

+1

-520 c-AMP

OTF PEA

+453

PEA

5

10

*

2

G C

0

β-actin

T-Lyn/ β-actin

p-Lyn/β-actin

C

52 10 3 Triptonide (nM)

D

Relative activity

Lyn/ β-actin

C β-actin 1.61.6

0

0

**

5 10 3 Triptonide (nM) 2

100 100

**

80 60 40 20 0 80

60

**

40

**

20

0

0 1

21

2.5 5 10 Triptonide (nM) 2

3

4

Figure 5 1.2 1.2

B

A

p-ERK/ T-ERK

C

p-Erk C T-ERK p-NF-kB T-NF-kB

1.01 0.8 0.8

**

0.4 0.2 00

p-p38

**

0.6 0.6

01

T-p38 p-AKT

T-AKT and p-AKT/ β-actin

C

T-AKT

C

p-CREB T-CREB p-STAT3 T-STAT3 β-actin Triptonide: (nM)

0

5

10

D C

3.5 3.5

p-p38/ T-p38

3.03 0

**

**

2.5 2.5 2.02 1.5 1.5 1.01 0.5 0.5 00

0 5 10 1 Triptonide 2 (nM) 3

22

52 10 3 Triptonide (nM)

1.4 T-AKT

1.2

p-AKT

1.0 0.8 0.6

** **

0.4 0.2 0

0

5 10 Triptonide (nM)

Figure 6

23

Table 1. Triptonide does not significantly affect the blood cell count and parameters in mice

Blood parameters

Control group

Triptonide group

P-value

WBC(109/L)

2.55±0.12

3.48±0.21

0.69

RBC(1012/L)

7.05±0.96

6.58±0.0.97

0.14

HGB(g/dL)

178.63±5.20

166.90±5.07

0.20

MCV(fl)

45.37±0.92

45.15±1.08

0.51

MCH(pg)

25.70±2.21

25.85±2.3

0.93

MCHC(g/dL)

566.50±10.26

575.55±10.73

0.87

RDW-SD(fl)

26.51±2.69

24.44±3.25

0.49

HCT(%)

0.32±0.04

0.30±0.05

0.13

PLT(109/L)

718.63±22.58

795.30±24.06

0.68

MPV(fl)

4.42±1.62

4.08±1.66

0.71

PDW(fl)

5.19±1.78

4.64±1.77

0.6

RDW-CV(%)

0.16±0.02

0.17±0.03

0.24

After the human B-lymphoma cell-xenografted NOD/SCID mice (n=8 each group) were treated with triptonide for 34 days, blood was collected and the blood parameters were measured by Hematology Analyzer. The data were shown as mean ± SE. WBC: white blood cell count, RBC: red blood cell, HGB: hemoglobin, MCV: mean corpuscular volume, MCH: mean corpuscular hemoglobin, MCHC: mean corpuscular hemoglobin concentration, RDW-SD: red blood cell distribution width-standard deviation, HCT: hematocrit, PLT: platelet count, MPV: mean platelet volume, PDW: platelet distribution width, RDW-CV: red blood cell volume distribution width.

24