In GC cell lines, MCT1, 2, and 4 were up-regulated with different expression levels; MCT1 and MCT4 were more widely expressed in GC cell lines compared with MCT2. Inhibition of MCTs by siRNA or AR-C155858 reduced cell viability and lactate uptake in GC cell lines. The effect of inhibition of MCTs on tumor growth was also confirmed in xenograft models. Furthermore, MCT inhibition in GC cells increased the sensitivity of cells to radiotherapy or chemotherapy. Compared with normal gastric tissue, no significant alterations of expression levels in tumors were identified for MCT1 and MCT2, whereas a significant increase in MCT4 expression was observed. Most importantly, MCT4 was highly overexpressed in malignant cells of acsites and its silencing resulted in reduced tumor cell proliferation and lactate uptake in malignant ascites. Our study suggests that MCT4 is a clinically relevant target in GC with peritoneal carcinomatosis.Monocarboxylate transporters (MCTs) play a major role in up-regulation of glycolysis and adaptation to acidosis. However, the role of MCTs in gastric cancer (GC) is not fully understood. We investigated the potential utilization of a new cancer therapy for GC. We characterized the expression patterns of the MCT isoforms 1, 2, and 4 and investigated the role of MCT in GC through  Gastric cancer (GC) is the fourth most common cancer and the second leading cause of cancer deaths globally . In advaIn rapidly growing cancer cells, oncogenes and hypoxia stimulate the glycolytic metabolism, which generates increased amounts of lactic and carbonic acids –8. To avin vitro and in vivo . Co. Co47]. Peritoneal carcinomatosis in advanced GC remains an intractable clinical problem despite advances in the treatment of GC. Peritoneal dissemination is the most frequent pattern of gastric cancer recurrence (33-50%) , 52, 53.Our results suggest that MCT4 plays a central role in tumor metabolism in GC with peritoneal carcinomatosis and targeting MCT4 in combination with chemotherapy could be a novel strategy in the treatment of GC.in vivo GC xenograft models and enhance the anti-tumor activity of radiotherapy or conventional chemotherapy. Fourth, for identifying the significant differences in MCT expression between normal tissue and tumor tissue, we compared the levels of MCT expression of normal gastric tissue, primary GC tissue, and PDCs derived from malignant ascites by using real-time PCR.The objective of this study was to assess whether the MCTs could be a therapeutic target for treatment of advanced GC patients. First, we examined the expression levels of MCTs including MCT1, 2, and 4 in human GC cell lines by reverse transcription polymerase chain reaction (RT-PCR) and western blot. Second, to investigate the functional role of MCTs in GC, we explored the effect of knockdown of MCTs on cell proliferation and lactate uptake. Third, we examined whether inhibition of MCTs could translate in This study was approved by the Samsung Medical Center Institutional Review Board  in accordance with the ethical principles of the Declaration of Helsinki and local guidelines. Written informed consent was obtained from all participating patients.We screened 16 established GC cell lines including 6 primary cell lines , 4 metastatic cell lines , and 6 ascite cell lines  in order to identify the cell lines that expressed high levels of MCT1, 2, and 4.Total cellular RNA was extracted using an RNeasy MiniKit (Qiagen) and treated with DNase I. One microgram of RNA was converted to cDNA using an Omniscript RT Kit (Qiagen). Real-time PCR was performed by using a Prism 7900HT Sequence Detection System (PE Applied Biosystems).For western blot analyses, antibodies against MCT1 , MCT2 , MCT4 , and β-actin  were used. The protocols used are described in the Supplementary Materials.Silencing of MCT expression was done with siRNA targeting MCT1, MCT2, and MCT4  using HiPerfect transfection reagents (Qiagen) according to the manufacturer's instructions.5 cells per 6-well plate, Corning) were transfected with siRNAs and incubated for 3 days. Cell proliferation following each treatment was compared with untreated cells. For the clonogenic assay, cells were transfected with siRNAs for 24 hours, irradiated with a 137Cs source , incubated for 14 days, and then colonies were counted. All these experiments are described in detail in the Supplementary Materials.To assess cell numbers, cells (1 × 1014C]-L-lactate (Amersham Biosciences) was expressed as counts per million per milligram of protein. The protocols used are described in the Supplementary Materials.For lactate uptake measurements, siRNA-transfected cells were equilibrated in buffered solution adjusted to pH 6. [in vivo, we implanted mice subcutaneously or intraperitoneally with GC cell lines and assigned the animals to two groups based on their treatment with PBS only or AR-C155858. All mice experiments were conducted in accordance with the Institute for Laboratory Animal Research Guide for the Care and Use of Laboratory Animals, and the protocols were approved by the appropriate Institutional Review Boards at Samsung Medical Center (Agreement- 20141211001). Xenograft models of GC are described in the Supplementary Materials.To determine whether an MCT inhibitor has antitumor effects Forty-five matched pairs of primary GC and normal gastric tissue and PDCs (N = 48) were collected at the Samsung Medical Center. All GC tumors and control tissues were confirmed by the hospital's clinical pathology department. To establish PDCs from metastatic GC patients with malignant effusion, those who were enrolled in the SMC Oncology Biomarker study (NCT#01831609) were screened for the expression of MCTs by western blot. Details of cell lines and cell cultures are provided in the Supplementary Materials.All patients provided written informed consent. This study was performed in accordance with the Declaration of Helsinki and was approved by the Institutional Review Board of Samsung Medical Center.MCT4 expression status in patients enrolled in the ARTIST phase III clinical trial [We examined al trial . All slit-test or one-way ANOVA using GraphPad Prism version 4.01 . For analysis of the MCT4 IHC cohort, SPSS statistical software version 20  was used. All comparisons were examined by the 2χ test or Fisher exact test. The Kaplan-Meier test was used to evaluate the survival rate and survival curves were compared by the log-rank test. A Cox proportional hazard model was applied for multivariate survival analysis. All tests were two-tailed, and P values < 0.05 were considered significant.Preclinical data were evaluated by a two-tailed


There is limited evidence assessing the effects of omega-3 polyunsaturated fatty acids (PUFAs) on oesophageal adenocarcinoma, both in vitro and in vivo. We evaluated the effects of the omega-3 PUFA and oxaliplatin on OE33 and OE19 cells.The two oesophageal cells were treated with Omegaven® (fish oil emulsion), EPA, DHA and oxaliplatin and incubated for up to 144 h.p = 0.001), DHA by 59% (p < 0.000) and Oxaliplatin by 77% (p < 0.000). For OE19 cells, the EPA reduced growth by 1% (p = 0.992), DHA by 26% (p = 0.019) and oxaliplatin by 76% (p < 0.000). For both cells, Omegaven® resulted in reduced cell growth at intermediate concentrations (20-40 μM) and increased cell growth at low (10 μM) and high (50 μM) concentrations. DHA, Omegaven® and oxaliplatin were associated with significant downregulation of VEGF and p53 protein, and upregulation of p21 protein. DHA, Omegaven® and Oxaliplatin also led to significant downregulation of the total ERK1/2 and Akt proteins.The following inhibitory effects were observed on OE33 cells: EPA reduced cell growth by 39%  had greater in vitro anti-cancer effects than EPA alone.The online version of this article (10.1186/s12944-018-0664-1) contains supplementary material, which is available to authorized users. There is an increasing interest in natural therapies with anti-inflammatory effects that exhibit anticancer benefit, including omega-3 PUFAs. The main omega-3 PUFA are eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) which have anti-inflammatory effects –3. Only Helicobacter pylori (H. pylori) related chronic gastritis and atrophy [H. pylori infection causes the induction of various pro-inflammatory cytokines such as TNF-α, Interleukin (IL) -1 and IL-6 [Several factors in the cancer microenvironment influence the carcinogenesis process, including the delivery of bioactive molecules, such as cytokines and growth factors that are responsible for increased cell proliferation, inhibition of apoptosis and induction of angiogenesis . In many atrophy . It is eand IL-6 . For theWu et al., treated gastric cancer cell lines with EPA and DHA, and found inhibited macrophage activated cell migration by down regulation of the matrix metalloproteinase 10 gene, and subsequent down regulation of extracellular signal-related kinase (ERK) . SlagsvoIn this exploratory study, we evaluated the effect of the four single treatments  and oxaliplatin) on OE33 and OE19 cell growth and expression of the following cytokines: IL-6, TNF-α and VEGF in the cell culture supernatant. In addition, we also evaluated expression of the following proteins p53, p21, Akt, ERK1/2 in the cell lysate.The two oesophageal cancer cell lines used were OE19 and OE33. OE19 is a human oesophageal cancer cell line derived from a 72 year old white male patient with moderately differentiated UICC stage 3 adenocarcinoma. The OE33 cancer cell line is derived from a 73 year old white female with UICC stage 2A lower oesophageal adenocarcinoma arising in a background of known Barrett’s metaplasia. These cell lines were purchased from Public Health England cell collection (The European Collection of Authenticated Cell Cultures).2. Both cell lines were cultured in RPMI 1640 medium  supplemented with 2 mM Glutamine and 10% foetal bovine serum (FBS).Cell lines were cultured as a monolayer at 37 °C and 5% COCell lines were passaged no more than 15 times following resuscitation from liquid nitrogen, to reduce the risk of phenotypic alterations. Passaging was undertaken once cells had reached approximately 80% confluence as follows: Cells were washed with 10 mL pre-warmed (37 °C) PBS once, followed by addition of 5 mL of 1X trypsin for 5 min at 37 °C for cell detachment. The trypsinisation process was halted following addition of an equivalent volume of RPMI media containing 10% FBS. Cells were pelleted at 400 x g, resuspended in fresh medium containing 10% FBS, and aliquoted appropriately into cell culture flasks as per experimental requirements.The treatments tested were EPA, DHA, Oxaliplatin , and Omegaven® . EPA and DHA stocks were prepared as 50 mM stocks dissolved in DMSO and oxaliplatin was prepared as a 50 mM stock dissolved in 5% dextrose. All treatments including the vehicle control, received equivalent volumes of DMSO or 5% dextrose. Omegaven® is a 10% fish oil lipid emulsion containing 1.25 to 2.82 g/100 ml EPA and 1.44 to 3.09 g/100 ml DHA as per the Omegaven® summary of product characteristics. The rationale for selecting Omegaven® was that it was commercially available, the omegaven® emulsion was also investigated over the same time period in a pilot clinical trial in patients with advanced oesophago-gastric cancer and the intention was to mirror the in vitro laboratory work with the clinical trial.OE33 and OE19 cell lines were grown in RPMI 1640 + 2 mM Glutamine + 10% foetal bovine serum (FBS) medium for 24 h, then the media was removed and replaced with medium containing 10 μM, 20 μM, 30 μM, 40 μM and 50 μM of EPA, DHA and Oxaliplatin treatment and in order to equate the Omegaven® emulsion mixture to treatment concentrations using the single agents, the emulsion was diluted in RPMI medium + 10% FCS via serial dilution to make treatments of approximately 10 μM, 20 μM, 30 μM, 40 μM and 50 μM of EPA and DHA. The cell lines were incubated for 72, 96, 120 or 144 h to determine the anti-proliferative effects. The cell culture supernatant was collected at 72 and 144 h and stored at − 80 °C for cytokine analysis. The cells were then harvested and counted.3 cells/well in 1 mL of RPMI 1640 medium. Cells were incubated for 24 h, and then the media was replaced with media containing the relevant treatments. Cells were then incubated for 72, 96, 120 and 144 h before counting; Each well was washed × 1 with 1 mL PBS and 0.5 mL of 1X trypsin added to each well, which was neutralised with 0.5 mL RPMI + 10% FBS once cells had detached. The well contents were transferred to coulter count cups containing 9 mL of isoton solution  and cells were counted using the Z2 particle analyser.Cell proliferation was undertaken using a Z2 particle size analyser  to count raw cell numbers; this was performed in both cell lines for comparison in triplicate. OE19 and OE33 cell lines were seeded into 24-well plates at a density of 2 X 10Cell culture media supernatants were collected at 72 and 144 h after incubation with treatment as described above, and stored at − 80 °C for analysis of IL-6, TNF-α, VEGF using an ELISA assay.5 cells per 75 cm flask and allowed to adhere overnight. Cells were then treated with EPA, DHA, Omegaven® or Oxaliplatin at concentrations of 10 μM and 50 μM and incubated for 72 and 96 h.The cell lines (OE19 and OE33) were seeded at 1.5 X 10After 72 and 96 h, all flasks were removed from the incubator and placed on ice. Cells were gently scraped using a sterile cell scraper, media containing cells was collected and cells pelleted . The supernatant was discarded, and the remaining cell pellet washed with 1 mL PBS. Cells were again pelleted, the PBS carefully aspirated and 100 μL of Roche Complete Lysis M  cell lysis buffer added to the cell pellet. The pellet was mixed by vigorous pipetting and the cells lysed on ice for 10 min. Cell debris was pelleted and the supernatant stored at − 80 °C for later analysis of total Akt, Erk1/2, p53 and p21 proteins.The data was not normally distributed hence all data were transformed to log 10. In order to identify whether there was any significant effect of each treatment, a two tailed t test was used to compare cell counts and all cytokine expression compared to the control.Growth inhibition of both cell lines was observed following treatment with EPA, DHA and oxaliplatin. The growth inhibition occurred in a time-, dose- and cell line dependent manner, with OE33 cells exhibiting more sensitivity to treatment than OE19 cells.p = 0.001), DHA reduced cell growth by 59% (p < 0.000) and Oxaliplatin reduced cell growth by 77% (p < 0.000). For OE19 cells, the EPA reduced growth by 1% (p = 0.992), DHA reduced growth by 26% (p = 0.019) and oxaliplatin reduced cell growth by 76% (p < 0.000). For both cell lines, the effects of Omegaven on cell growth did not follow a linear pattern, but more a “U-shaped” curve, with increased cell growth at low (10 μM) and high (50 μM) concentrations, and decreased cell growth at intermediate (20–40 μM) concentrations, see Additional file The following inhibitory effects were observed on OE33 cells: Compared to control, EPA reduced cell growth by 39% p = 0.00, DHA red® and oxaliplatin treatment was associated with significant down regulation of VEGF expression compared to control. A similar concentration of EPA was associated with an upregulation effect  significantly downregulated Akt expression in both cell lines after 96 h Fig. . All tre0 μM signIt has been reported in four previous studies on gastric adenocarcinoma cell lines that omega-3 PUFAs (mainly EPA and DHA) present in fish oil are associated with anti-proliferative effects , 10–12, We have shown for the first time that EPA, DHA, and oxalipatin have anti-proliferative effects on OE33 and OE19 cell lines. There was a more pronounced dose response effect with DHA and oxaliplatin than with EPA. Oxaliplatin as a single treatment or coupled with EPA has been previously investigated in colorectal cancer cell lines , 16. TheThis study differs from previous studies that investigated omega-3 PUFAs in that we used EPA and DHA as a single treatment and in combination in the form of a fish oil emulsion (Omegaven®) in oesophageal adenocarcinoma cell lines for the first time. At low 10 μM) and high (50 μM) concentrations of Omegaven®, treatment resulted in a significant increase in cell number for both OE19 and OE33 cell lines, this effect was lost at intermediate concentrations of 20 μM and 30 μM as shown in the Additional file 0 μM and Granci et al., investigated the effects of Omegaven®  in combination with oxaliplatin in colorectal cancer HT-29 cells, with this combination associated with further significant reduction of cell viability when compared to oxaliplatin alone .Omegaven® emulsion has various other substances such as preservatives, stabilisers and also other fatty acids including sodium oleate (monounsaturated fatty acid). The latter might theoretically have altered the growth characteristics of the oesophageal cancer cell lines as it had previously reported to increase breast cell line proliferation and decrease apoptosis , 21. In In colonic cancer cells, both EPA and DHA (10–30 μM) were associated with inhibition of VEGF expression . In vivoIn the current series of experiments, the most profound effects on cytokine production were on VEGF. The treatment of OE33 cells with DHA, Omegaven® and oxaliplatin in this experiment, showed significant decreases in VEGF expression after prolonged treatment of 144 h  was examined and we found that EPA treatment was associated with a significant increase in IL-6 and TNF-α expression as early as 96 h of treatment in OE33 cells and 120 h in OE19 cells. DHA, Omegaven® and oxaliplatin led to dose- and time dependent biphasic changes in OE33 cells. This sort of inconsistent low dose vs high dose effect appears to be a common finding in response to a variety of dietary agents and has previously been reported by Lavigne et al. in 2008, while investigating the effects of isoflavones (Genistein) in breast cancer cell lines . Cai H eIt had been previously reported that omega-3 PUFAs inhibited the activation of NF-kB, which normally regulates the Interleukin-6 and TNF-α expression , 27. The® and oxaliplatin treatments in both cells were associated with significant downregulation of p53 and upregulation of p21 protein, but this was not the case for EPA  forms of these proteins. Future work should assess the effects of treatment on protein phosphorylation for both Akt and ERK 1/2, which would give a more comprehensive indication of which up-stream pathways are directly implicated in regulating PUFA efficacy.We have demonstrated that DHA, Omegaven® and oxaliplatin were associated with downregulation of p53 and VEGF in two oesophageal adenocarcinoma cell lines. Of the PUFAs studied, DHA alone or in combination (Omegaven®) had greater in vitro anti-cancer effects than EPA alone. It appeared to exert its anti-cancer effects via multiple different pathways. Further, the magnitude of the anti-proliferative effect observed for DHA was similar to that observed with oxaliplatin in these oesophageal cancer cell lines. We speculate that it could be used as an adjunct to chemotherapy for oesophageal adenocarcinoma to enhance treatment efficacy.


Epigenetic abnormalities play important roles in nasopharyngeal cancer (NPC), however, the epigenetic changes associated with abnormal cell proliferation remain unclear.ZNF671 in NPC tissues and cell lines by bisulfite pyrosequencing. We evaluated zinc finger protein 671 (ZNF671) expression in NPC cell lines and clinical tissues using real-time PCR and western blotting. Then, we established NPC cell lines that stably overexpressed ZNF671 and knocked down ZNF671 expression to explore its function in NPC in vitro and in vivo. Additionally, we investigated the potential mechanism of ZNF671 by identifying the mitotic spindle and G2/M checkpoint pathways pathway downstream genes using gene set enrichment analysis, flow cytometry and western blotting.We detected epigenetic change of ZNF671 was hypermethylated in NPC tissues and cell lines. The mRNA and protein expression of ZNF671 was down-regulated in NPC tissues and cell lines and the mRNA expression could be upregulated after the demethylation agent 5-aza-2′-deoxycytidine treatment. Overexpression of ZNF671 suppressed NPC cell proliferation and colony formation in vitro; silencing ZNF671 using a siRNA had the opposite effects. Additionally, overexpression of ZNF671 reduced the tumorigenicity of NPC cells in xenograft model in vivo. The mechanism study determined that overexpressing ZNF671 induced S phase arrest in NPC cells by upregulating p21 and downregulating cyclin D1 and c-myc.Epigenetic mediated zinc finger protein 671 downregulation promotes cell proliferation and enhances tumorigenicity by inhibiting cell cycle arrest in NPC, which may represent a novel potential therapeutic target.The online version of this article (10.1186/s13046-017-0621-2) contains supplementary material, which is available to authorized users. Nasopharyngeal carcinoma (NPC) is the most common head and neck cancer in Southern China and Southeast Asia . AlthougUncontrolled proliferation is a pathological characteristic of cancer cells. Protein kinase complexes composed of cyclins and cyclin-dependent kinases (CDKs) determine the progression of cells through the cell cycle. Cyclins function as the regulatory subunit and CDKs function as the catalytic subunit of the activated heterodimer complexes, which orchestrate coordinated entry into the S phase of the cell cycle . DysreguZNF671), which contains C2H2-type zinc fingers (ZFs) and a Krüppel associated box (KRAB) domain, is a member of the KRAB-ZFP family of mammalian transcriptional repressors [ZNF671 is epigenetically silenced by DNA methylation and functions as a tumor suppressor in multiple carcinomas [ZNF671 in NPC.DNA methylation is a critical epigenetic modification involved in regulation of gene expression . Dysregupressors , 13 thatpressors , 15. Recrcinomas –18. HoweZNF671 is downregulated and the ZNF671 promoter is hypermethylated in NPC cell lines and tissues. Overexpression of ZNF671 suppressed, while silencing ZNF671 promoted, NPC cell proliferation and colony formation in vitro and tumorigenicity in vivo. Further studies demonstrated overexpression of ZNF671 inhibited NPC cell proliferation and tumorigenicity by inducing S phase cell cycle arrest.Here, we report that Human NPC cell lines  were cultured in RPMI-1640  supplemented with 5% fetal bovine serum (FBS) . Human immortalized nasopharyngeal epithelial cell line  were cultured in keratinocyte serum-free medium (Invitrogen) supplemented with bovine pituitary extract . 293 T cells were obtained from the ATCC  and maintained in DMEM (Invitrogen) supplemented with 10% FBS. Four freshly frozen NPC samples and four normal nasopharyngeal epithelium samples were collected from patients undergoing biopsy at Sun Yat-sen University Cancer Center.ZNF671 forward, 5′- GACTTAGACCTGGTTGTTGG -3′ and reverse, 5′- GTATTTAGCCAGGTGTAAGGT-3′. GAPDH was used as control for normalization.Total RNA was isolated from NPC cell lines using TRIzol Reagent (Invitrogen) following the manufacturer’s instructions, cDNA was synthesized using M-MLV reverse transcriptase  and amplified with Platinum SYBR Green qPCR SuperMix-UDG reagents (Invitrogen) using the CFX96 sequence detection system  with the following primers: ZNF671 , anti-cyclin D1 , anti-c-myc  or anti-p21  antibodies overnight at 4 °C, followed by species-matched secondary antibodies. Bands were detected using enhanced chemiluminescence.RIPA lysis buffer  was used to isolate proteins and the Bradford method, to determine protein concentrations. Proteins (20 μg) were separated by SDS-polyacrylamide gel electrophoresis , transferred onto PVDF membranes  and incubated with primary anti-NPC cell lines were treated with or without 10 μmol/L 5-aza-2′-deoxycytidine  for 72 h, with the drug/media replaced every 24 h. DNA was isolated using the EZ1 DNA Tissue Kit , then 1–2 μg DNA was treated with sodium bisulfite using the EpiTect Bisulfite kit (Qiagen) according to the manufacturer’s instructions. Bisulfite pyrosequencing primers were designed using PyroMark Assay Design Software 2.0 (Qiagen), and were: PCR forward primer: 5′-GAATTTAGGTTAGGGATAGTTTGAT-3′ (F); PCR reverse primer: 5′-CCAAAAAAAAAATATTTCAATACC-3′ (R); sequencing primer: 5′-GG ATAGTTTGA TAGAAATAAAATG-3′(S). The PyroMark Q96 System (Qiagen) was used for the sequencing reactions and to quantify methylation.ZNF671-HA or pSin-EF2-puro-vector plasmids were obtained from Land. Hua Gene Biosciences ; pSin-EF2-puro-Vector plasmid was used as a control. Stably transfected cells were selected using puromycin and confirmed using RT-PCR. SiRNAs targeting ZNF671 were obtained from GenePharma Co., Ltd. ; siRNA #1 targets ZNF671-Homo-626 cDNA  and siRNA #2 targets ZNF671-Homo-279 cDNA .The pSin-EF2-puro-3) were seeded into 96-well plates, incubated for 0–4 days, stained with CCK-8 , and absorbance values were determined at 450 nm using a spectrophotometer. For the colony formation assay, cells (0.3 × 103) were seeded into 6-well plates, cultured for 2 weeks and the colonies were fixed in methanol, stained with crystal violet and counted.For the CCK-8 assay, cells (1 × 105) were seeded into 6-well plates, cultured for 24 h, serum-starved for 24 h to synchronize cells at the G1/S checkpoint, trypsinized, washed with ice-cold PBS, fixed in 70% ethanol, and stored at −20 °C until analysis. Before staining, cells were gently resuspended in cold PBS and RNase A was added into cell suspension tube incubated at 37 °C for 30 min, followed by incubation with propidium iodide (PI) (Beyotime) for 20 min at room temperature. The fluorescence intensity of the cells was analyzed by flow cytometry .Cells (2 × 10ZNF671 cells (1 × 106) were subcutaneously inoculated into the dorsal flank. Tumor size was measured every 3 days and tumor volumes were calculated using the equation: volume = D × d2 × π/6, where D and d represent the longest and shortest diameters, respectively. All animal research was conducted in accordance with the detailed rules approved by the Animal Care and Use Ethnic Committee of Sun Yat-sen University Cancer Center and all efforts were made to minimize animal suffering.BALB/c-nu mice (4–6 weeks old) were purchased from Charles River Laboratories , and CNE2-vector or CNE2-www.broadinstitute.org/gsea/) was used to identify KEGG pathways  that show an overrepresentation of up- or downregulated genes between ZNF671 high expression (n = 15) and ZNF671 low expression (n = 16) in GSE12452. Briefly, an enrichment score was calculated for each gene set  by ranking each gene by their expression difference using Kolmogorov-Smirnov statistic, computing a cumulative sum of each ranked in each gene set, and recording the maximum deviation from zero as the enrichment score.The GSEA software tool .Statistical analyses were performed using SPSS 17.0 . All data shown are representative of at least three independent experiments, and values are expressed as the mean ± SD. Differences between two groups were analyzed using the two-tailed unpaired Student’s ZNF671 was detected by bisulfite pyrosequencing analysis in other NPC (n = 8) and normal tissues (n = 8). The CpG islands and region selected for bisulfite pyrosequencing in the ZNF671 promoter region are shown in Fig. ZNF671 (cg11977686) in NPC tissues were significantly increased compared with normal tissues  methylation levels in the NPC cell lines  were also increased compared with human immortalized normal nasopharyngeal epithelial cell line (NP69) . These results indicate that ZNF671 promoter is hypermethylated in NPC.To confirm our previous methylation data (GSE52068) . Furthermore, western blotting showed ZNF671 protein expression was downregulated in both the NPC cell lines and freshly frozen NPC tissues (n = 4) compared to normal samples (n = 4) . To determine whether the downregulation of ZNF671 results from its promoter hypermethylation, immortalized normal nasopharyngeal epithelial cell line and NPC cell lines were treated with or without the demethylation drug 5-aza-2′-deoxycytidine . The ZNF671 methylation level were substantially decreased , while the ZNF671 mRNA were significantly increased  in NPC cell lines compared with immortalized normal nasopharyngeal epithelial cell. The findings suggest that ZNF671 is downregulated in NPC and the downregulation of ZNF671 is associated with the hypermethylation of ZNF671 in NPC.To know the association between lls Fig. . AnalysiZNF671 on proliferation and metastasis in NPC, we subjected CNE2 and 5-8F cells stably overexpressing ZNF671 or the control vector to the CCK8, colony formation and Transwell migration and invasion assays. As shown in Fig. ZNF671 mRNA and protein level was obviously elevated after stably overexpressing ZNF671 in NPC cells. The CCK8 assay demonstrated that the cell viability of CNE2 and 5-8F cells stably overexpressing ZNF671 remarkably was much slower than that of cells expressing vector plasmid  in the GEO database to identify pathways potentially linked to ZNF671. As shown in Fig. ZNF671-high expressing tumors; conversely, these pathways were not enriched in ZNF671-low expressing tumors.To further investigate the mechanism by which ZNF671 significantly decreased the percentage of cells in the G2/M phase  and increased the proportion of cells in the S phase . Furthermore, bioinformatics analysis indicated that ZNF671 is involved in regulation of the cycle-related proteins cyclin D1 and p21. Overexpression of ZNF671 decreased the protein levels of cyclin D1 and c-myc, and increased p21  Methylation levels of ZNF671 in Normal (n = 24) and NPC (n = 24) tissues from the genome-wide methylation microarray data. Mean ± ± SD; Student’s t-tests. (B) Bisulfite pyrosequencing analysis of the ZNF671 promoter region in NP69 and NPC  cell lines. Red words: CG site of cg11977686. *P < 0.05, **P < 0.01 vs. control, Student’s t-test. (TIFF 831 kb)Additional file 2: Figure S2.ZNF671 is hypermethylated in NPC cells. Bisulfite pyrosequencing analysis of the ZNF671 promoter region in NP69 and NPC  cell lines following treatment with DAC. Red words: CG site of cg11977686. *P < 0.05, **P < 0.01 vs. control, Student’s t-test. (TIFF 861 kb)Additional file 3: Figure S3.ZNF671 has no effect on affect NPC migratory and invasive ability. (A) Migration ability was measured using a wound healing assay (200 ×) and (B) Transwell assay with Matrigel (200 ×) in CNE2 and SUNE1 cells with the vector or ZNF671 overexpression. Scale bar: 100 μm; data are mean ± SD. *P < 0.05, **P < 0.01 vs. control, Student’s t-test. (TIFF 1066 kb)


No standard of care is established for plasmablastic lymphoma (PBL) and prognosis remains extremely poor, given that patients relapse early after chemotherapy and display resistance to commonly applied cytostatic drugs.We report a case of nodal, HIV-unrelated PBL in a patient who achieved and maintained a very long lasting complete remission after an intensive therapy consisting consisting of thalidomide plus dexamethasone followed by a consolidation with double autologous stem cell transplantation. Our approach was based on the full application of a standard multiple myeloma treatment and, to the best of our knowledge, it represents the only reported experience so far. This treatment was overall well tolerated.Multiple myeloma-like treatment may represent a possible alternative to intensive lymphoma-directed therapies. Plasmablastic lymphoma (PBL) is a rare and highly aggressive subtype of diffuse large B-cell lymphoma, characterized by diffuse proliferation of large neoplastic cells which resemble B-cell immunoblasts but have a plasma cell immunophenotype . No stanA 46-year old Italian female presented in June 2007 with 3 enlarged lymph nodes in her left groin without any systemic symptoms. She had no significant comorbidities and no underlying immunosuppression. An excisional biopsy showed diffuse proliferation of large lymphoid cells with several interspersed macrophages, which imparted a “starry-sky” appearance to the infiltrate Fig. . ImmunohOn admission, laboratory data were the following: white blood cells 9500/μL, hemoglobin 12.5 g/dL, platelets 313,000/μL; lactate dehydrogenase, calcium, serum proteins and immunoglobulin levels were all within normal ranges; protein electrophoresis and immunofixation showed no monoclonal component. Serology for human immunodeficiency virus (HIV) was negative.From July to September 2007, the patient received 3 cycles of thalidomide and dexamethasone: thalidomide was given at the dose of 100 mg/day and dexamethasone at the dose of 40 mg/day on days 1–4 and 9–12 of each cycle. The rationale for the use of thalidomide in PBL relied upon the notion that increased angiogenesis plays a major role in aggressive lymphomas, as well as in MM [2) and granulocyte-colony stimulating factor (G-CSF): 7.2 × 106 CD34+/kg were harvested after a single apheresis. Following this procedure, the patient went back on thalidomide-dexamethasone for 3 more months, then she underwent an autologous stem cell transplantation (ASCT), conditioned with melphalan, 200 mg/m2. 2.5 × 106 CD34+/kg PBSC were reinfused. The PET scan performed one month after ASCT confirmed a CR. In light of the good response achieved so far, it was decided to proceed with a second ASCT. The patient again received thalidomide-dexamethasone for 3 months, then 2.5 × 106 CD34+/kg PBSC were reinfused 2 days after melphalan conditioning. A CR was clinically and metabolically confirmed by PET scan after the second ASCT. The patient is now healthy and in durable CR after at least 10 years.Afterwards, the patient received maintenance therapy with thalidomide for 2 months, followed by peripheral blood stem cell (PBSC) mobilization achieved by the administration of high dose cyclophosphamide  and has been performed in accordance with the ethical standards of the Declaration of Helsinki. Patient provided a written informed consent to publish her data.PBL is a highly aggressive lymphoma strongly associated with immunosuppression, in particular with HIV infection . NeverthMYC occur in up to 47% of HIV-related PBL cases and end up with overcoming the regulatory effects of its repressors Bcl-6 and BLIMP-1, thus allowing cells to proliferate without control. Moreover, MYC translocations are thought to contribute to the plasmablastic morphology and to a worse prognosis [The pathogenesis of PBL is currently unclear. The neoplastic cell derives from a post-germinal centre activated B-cell that has already undergone somatic hypermutation and class switching recombination and is in the process of becoming a plasma cell . Mutatiorognosis . The HIVMaking a diagnosis of PBL is always challenging: the immunophenotype resembles that of plasma cell neoplasms, as PBL cells typically lack expression of B-cell markers  and show little to no expression of leukocyte common antigen CD45, whereas the plasma cell markers CD79a, IRF4/MUM-1, BLIMP-1, CD38 and CD138 are usually highly expressed. The proliferation rate is generally high, with Ki-67 expression higher than 60% [Given its rarity and peculiar features, no standard of care can be established for PBL patients, whose prognosis remains very poor. Early relapses and subsequent chemotherapy resistance characterize the clinical course of the disease. A large literature review of 248 cases of PBL, 50% of which had received cyclophosphamide, doxorubicin, vincristine, prednisone (CHOP) or CHOP-like regimens, while 23% had been treated with more intensive regimens, such etoposide, vincristine, doxorubicin, cyclophosphamide, prednisone (EPOCH), hyperfractionated cyclophosphamide, vincristine, doxorubicin, dexamethasone (hyper-CVAD) and cyclophosphamide, vincristine, doxorubicin, methotrexate alternating with ifosfamide, etoposide, cytarabine (CODOX-M/IVAC), showed an overall response rate to chemotherapy of 77%, with 46% of patients achieving a CR and 31% a partial response [On the basis of the plasmacytic differentiation of this lymphoma, given that a plasmablast is an activated B-cell undergoing the process of becoming a plasma cell, PBL is ontogenetically collocated between an aggressive post-germinal centre B-cell non-Hodgkin lymphoma and MM. For this reason, the use of antimyeloma agents – like bortezomib, thalidomide or lenalidomide - may have a sound rationale, although the experience with these agents is rather limited. More specifically, they have been predominantly combined with more standard lymphoma-directed regimens, such as CHOP or EPOCH –15, rath


NF1 gene deficiency leads to Ras hyperactivation causing the subsequent activation of the AKT/mTOR and Raf/MEK/ERK pathways and inducing multiple cellular responses including cell proliferation. In this study, three NF1-null MPNST-derived cell lines , STS26T sporadic MPNST cell line and PNF-derived primary Schwann cells were used to test responses to AZD8055, an ATP-competitive “active-site” mTOR inhibitor. In contrast to rapamycin treatment which only partially affected mTORC1 signaling, AZD8055 induced a strong inhibition of mTORC1 and mTORC2 signaling in MPNST-derived cell lines and PNF-derived Schwann cells. AZD8055 induced full blockade of mTORC1 leading to an efficient decrease of global protein synthesis. A higher cytotoxic effect was observed with AZD8055 compared to rapamycin in the NF1-null MPNST-derived cell lines with IC50 ranging from 70 to 140 nM and antiproliferative effect was confirmed in PNF-derived Schwann cells. Cell migration was impaired by AZD8055 treatment and cell cycle analysis showed a G0/G1 arrest. Combined effects of AZD8055 and PD0325901 MEK inhibitor as well as BRD4 (BromoDomain-containing protein 4) inhibitors showed a synergistic antiproliferative effect. These data suggest that NF1-associated peripheral nerve sheath tumors are an ideal target for AZD8055 as a single molecule or in combined therapies.Approximately 30-50% of individuals with Neurofibromatosis type 1 develop benign peripheral nerve sheath tumors, called plexiform neurofibromas (PNFs). PNFs can undergo malignant transformation to highly metastatic malignant peripheral nerve sheath tumors (MPNSTs) in 5-10% of NF1 patients, with poor prognosis. No effective systemic therapy is currently available for unresectable tumors. In tumors, the  Neurofibromatosis type 1  is an autosomal disorder with a worldwide birth incidence of one in 2500. Approximately 30-50% of individuals with NF1 develop benign peripheral nerve sheath tumors, called plexiform neurofibromas (PNFs) that are usually congenital or appear in early childhood . PNFs caNeurofibromin 1 (NF1) encoding neurofibromin, a negative regulator of RAS (rat sarcoma) proteins. NF1 loss of heterozygosity was found in Schwann cells isolated from PNFs and MPNSTs .–18.NF1 lCDKN2A/B .NF1 locus were found in DNAs from 3/8 of the PNF-derived primary Schwann cells: PNF1, PNF2, and PNF8  or rapamycin (10nM) respectively in 90.8 cell lines, and a decrease of 82% vs 12% when using AZD8055 (100nM) or rapamycin (10nM) respectively in STS26T cell lines . This inhibition correlates to the concentration of AZD8055 required to fully inhibit the phosphorylation of 4E-BP1 on S65 in all cell lines tested . In contrast, cell cycle progression was impaired. After 24h of treatment, AZD8055 significantly inhibited MPNST-derived cell lines proliferation PD901 = 3.7±0.8μM and IC50PD901 = 6.8±2.3μM, respectively) whereas STS26T and 90-8 were not sensitive . To a lesser extent, 96-2 cell line was also sensitive to JQ1 (IC50JQ1 = 66±22μM). In contrast, 88-14 and STS26T cell lines were JQ1-resistant  and, to a lesser extent, 88-14 and 96-2 cell lines were also . In contrast, STS26T was OTX015-resistant  and, to a lesser extent, 96-2 and 88-14 cell lines were also (IC50I-BET-762 = 66±32μM and IC50I-BET-762 = 95±10μM respectively). In contrast, STS26T was I-BET-762-resistant . PNF3 derived primary Schwann cells did not response to JQ1 (not shown). Due to little or no response of PNF-derived primary Schwann cells to JQ1, IC50 were only assessed with JQ1 BET bromodomain inhibitor.Dose-response analyzes after 48 hours of continuous exposure of the three JQ1, OTX015, I-BET-762/GSK525762 BET bromodomain inhibitors were performed in STS26T, 90-8, 88-14, and 96-2 MPNST-derived cell lines. 90-8 cell line was sensitive to JQ1  supporting the relevance of combined mTOR kinase inhibitor AZD8055 and BET bromodomain inhibitors therapies. To a lesser extent, 96-2 cell line was also sensitive to BET bromodomain inhibitors and we confirmed the synergistic effect of AZD8055 combined to BET bromodomain inhibitors on this second cell line  showed antitumor activity on MPNST cell lines in vitro. mTOR inhibitors have been both characterized as leading to cell cycle arrest by suppressing cyclin D1 expression, inducing apoptosis, and inhibiting cell proliferation and angiogenesis, and shown to repress cell motility and invasion  Leucine assay. Cells were incubated for 2h at 105 cells/mL in a low leucine medium  then labelled for 1h with [3H] leucine . The amount of radioactivity incorporated into proteins was determined by trichloroacetic acid precipitation.The global protein synthesis was assessed by [Hp < 0.05, p < 0.01, p < 0.001 respectively.Data were expressed as mean values and SD. Statistical significance of differences observed between experimental groups was determined using Student's t test. *, **, *** mean


Plasmablastic lymphoma (PBL) is a rare, highly aggressive subtype of non-Hodgkin lymphoma with plasma-cell differentiation occurring typically in immune-suppressed patients such as those with AIDS. This study reports the establishment and characterization of a new cell line, PBL-1, derived from a patient with AIDS-associated PBL. Morphological assessment of PBL-1 indicated plasma-cell differentiation with a CD20(−) CD38(+) CD138(+) immunophenotype and IgH/c-myc translocation. The cell line harbours Epstein-Barr virus, but a 52.7-kbp length defect was identified in its genome, resulting in no expression of viral microRNAs encoded in the BamHI-A Rightward Transcript region. Importantly, supplementation of culture medium with >5 ng/mL of interleukin-6 (IL-6) was required for PBL-1 growth. Starvation of IL-6 or addition of tocilizumab, an inhibitory antibody for the IL-6 receptor, induced apoptosis of PBL-1. Transduction of IL-6 into PBL-1 by lentivirus vector induced autologous growth without IL-6 supplementation of culture medium. These data indicate the IL-6 dependency of PBL-1 for proliferation and survival. mTOR inhibitors induced cell death effectively, suggesting mTOR in the IL-6 signalling pathway is a potential therapeutic target for PBL. This established PBL cell line will be a useful tool to further understand the pathophysiology of PBL and aid the future development of PBL treatment. The incidence of PBL has been estimated as 2–8% of all cases of AIDS-related lymphomas4. Histologically, PBL cells have a large, round-to-irregular nucleus with a prominent nucleoli and perinuclear halo, as well as a variable amount of cytoplasm that is deeply basophilic2. PBL cells do not express an immunophenotype typical for B-cell  or T-cell  markers. Instead, several markers of lymphocyte activation  and plasma-cell differentiation  are usually detected.Plasmablastic lymphoma (PBL) is a rare distinct clinicopathological entity of large B-cell malignant lymphoma, occurring typically in HIV-1 infected or other immunodeficient patients and is generally associated with Epstein–Barr virus (EBV) infection6, suggesting it might play an important role in the tumorigenesis of PBL. EBV is a member of the oncogenic human gamma-herpesvirus family, and has been detected in Hodgkin lymphoma, Burkitt lymphoma, nasopharyngeal carcinoma, post-transplantation lymphoproliferative disorder, and gastric cancer. Approximately 40% of AIDS-related lymphoma cases are positive for EBV7. In PBL cases, EBV has been detected as Latency I, expressing EBV-encoded small RNAs (EBERs) and EBV-encoded nuclear antigen 1 (EBNA-1). Although integration of EBV in the host chromosome was observed in several cell lines established from EBV-associated lymphoma with persistent infection13, there has been no report of an EBV integrated case of PBL.EBV has been detected in 66–74% of PBL cases14. Currently, a combination of antiretroviral therapy16 with a CHOP -like regimen or more aggressive regimen including CODOX-M/IVAC , dose-adjusted EPOCH , or HyperCVAD  are considered first-line therapies18. Recently, cases treated with chemotherapy in combination with bortezomib, a proteasome inhibitor, have been reported24.The prognosis of PBL is very poor, and a standard therapy for PBL has not been establishedImmortalized cell lines are useful tools to investigate disease pathogenesis and to evaluate the effects of therapeutic agents. To the best of our knowledge, there has been no report describing a well-characterized PBL cell line established from typical PBL cases. In this study, we established a PBL cell line from a case of AIDS-associated PBL, and demonstrated its interleukin 6 (IL-6)-dependent growth. In addition, the treatment effect of therapeutic agents was investigated.2. After several passages, autologous growth was observed. However, when the ascites was removed from the culture medium, the cells did not grow and became apoptotic. Therefore, we speculated that certain growth factor(s) were required for cell growth. Cytokines in the patient’s ascites and the supplemented cultured medium were analysed by Luminex. A high amount of IL-6 was detected in the ascites , 10 ng/mL insulin, and 10 ng/mL of transferrin at 37 °C with 5% COIn situ hybridization demonstrated that PBL-1 were positive for EBER-1  with a triple colour-dual fusion probe of IgH and c-myc revealed that PBL-1 had an IgH/c-myc rearrangement , whereas there were no bcl-2 or bcl-6 gene splits (data not shown). PCR and sequence analysis identified a break point in the IgH/c-myc rearrangement that was upstream of exon 1 of c-myc and downstream of the IgH regulatory region , LMP-2A, and LMP-2B mRNA, indicating that the EBV status in PBL-1 was Latency I   3 pathway28. Flow cytometry analysis revealed the expression of gp80 and gp130 in PBL-1  Fig. . The XTTmL) Fig. . WesternmL) Fig. . Flow cymL) Fig. , and BrdmL) Fig. . IL-6 bi26. To verify the effect of IL-6 on STAT3 in PBL-1, we performed western blotting in the presence or absence of IL-6. While p-STAT3 was abundant in total cell lysates of PBL-1 in the presence of IL-6 (5 ng/mL), the dephosphorylation of p-STAT3 was induced within 1 h after the starvation of IL-6  and p70S6K and the dephosphorylation of 4E-BP-1, which are factors downstream of the mTOR pathway  inhibitors. The XTT assay showed that these drugs also inhibited the mitochondrial metabolic activity of PBL-1 in a dose-dependent manner  method. The combination of bortezomib and tocilizumab  MUM-1 (+) and CD20 (−), as reflected in PBL. The immunophenotype of PBL is similar to that of multiple myeloma except for those with EBV infection32. The established cell line, PBL-1, had an immunophenotype typical of plasmablasts, with morphological features that resembled plasmacytic tumours. To the best of our knowledge, there have been two previous reports of the establishment and characterization of cell lines from this distinct tumour34, and one report of a xenograft model of EBV-negative-PBL35. A study of two EBV-positive cell lines, SPIBL-II and III, established by Friis et al. did not report the immunophenotype or culture method used including IL-6-dependency33. The SPIBL-II and III cell lines were established from the pleural effusion of an HIV-positive case of plasmablastic lymphoma. Considering that PBL occurred in the ascites as an effusion lymphoma in our case, such an effusion form of PBL that is very rare might contribute to the establishment of a cell line. Another two PBL cell lines, KY-1 and KY-2, reported by Matsuki et al. were established from a immunocompetent patient without HIV infection, and were CD20 (−), CD138 (+), VS38c (+), EMA (+), but CD38 (−) and EBV (−)34. IL-6-dependency in the culture medium was not described for these reported cell lines, although IL-6 might be associated with the establishment of these cell lines in a xenograft model35.The cell origin of PBL is thought to be the plasmablast27. Starvation of IL-6 in PBL-1 led to the dephosphorylation of p-STAT3 and the phosphorylation of Akt, PDK1, and p70 within 1 h; however, p-Akt, p-PDK1, and p-p70 were dephosphorylated within 24 h  were effective at inducing apoptosis in PBL-1. Everolimus, an mTOR inhibitor, is an immunosuppressive drug used for the treatment of breast cancer, neuroendocrine tumours, and kidney cancer; however, there has been no report on the therapeutic use of everolimus for PBL. Inhibitors of the mTOR pathway such as everolimus or rapamycin were more effective on PBL-1 than inhibitors to other factors downstream of IL-6 signalling such as the Jak/Stat pathway. This suggests that mTOR-associated factors play an important role in the IL-6 signalling pathway in PBL-1, and that IL-6 signalling pathways, especially mTOR, are a potential therapeutic target for PBL. Furthermore, in this study, bortezomib, a proteasome inhibitor used for the therapy of multiple myeloma, also exhibited a cytotoxic effect on PBL-1. This finding may indicate that PBL-1 has similar pharmacological responses to multiple myeloma. A combination of HDAC and proteasome inhibitors enhanced apoptosis in some lymphomas, multiple myeloma, EBV-associated tumours and cell linesin situ hybridization. These results led to a diagnosis of AIDS-related PBL. Six courses of CHOP chemotherapy induced a remission, but the patient eventually succumbed to the recurrence of disease.A 60-year-old Japanese male patient with HIV-1 infection was admitted with fever, weight loss, and ascites. His blood CD4 (+) cell counts were 316/μL. Flow cytometry analysis indicated the presence of CD19 (−), CD20 (−), CD38 (+) cells in the ascites. Positron emission transfer imaging revealed a large tumour in the transversus colon, and its biopsied specimen showed the infiltration of tumour cells similar to those in the ascites. Tumour cells were CD19 (−), CD20 (−), CD79a (−), CD38 (+), CD138 (+), CD30 (−), EBV- LMP-1 (−), EBV-EBNA-2 (−), KSHV-latency-associated nuclear antigen 1 (−), CD3 (−) CD5 (−), CD10 (−), BCL6 (−), MUM1 (+), and >95% Ki67 (MIB-1) (+) by immunohistochemistry, and EBER-1 (+) by 2. Supplementation of insulin and transferrin was gradually reduced during passages. Finally, cells were grown in RPMI 1640 with 5 ng/mL of IL-6  and 10% FBS. Single cell cloning was performed using a limiting dilution method in IL-6 containing medium. For the IL-6 starvation study, cells were centrifuged, washed twice with phosphate buffered saline (PBS), and cultured in RPMI 1640 supplemented with 10% FBS without IL-6. EBV-positive human cell lines (Raji and LCL), B95.8, an EBV-positive marmoset cell line, and EBV-negative cell lines  were cultured in RPMI 1640 medium with 10% FBS. Cells were stained with trypan blue and cell viability was measured using a TC10 automated cell counter .To establish a new cell line, tumour cells were collected from the patient ascites and cultured in RPMI 1640 culture medium supplemented with 10% patient ascites, 10% FBS, 10 ng/mL of insulin , and 10 ng/mL of transferrin (Sigma-Aldrich) at 37 °C with 5% CO4 cells per spot. The samples were incubated with mouse monoclonal antibodies to CD20, CD38, CD138, and LMP-1 of EBV  diluted 1:100 with Block Ace  for 1 h at room temperature, followed by Alexa Fluor 488-conjugated goat anti-mouse IgG (H+L)  diluted 1:500 with PBS for 1 h at room temperature. Nuclear staining was performed with 1 μg/mL 4′,6-diamidino-2-phenylindole. Images were captured with a fluorescence microscope .Suspended cells were centrifuged onto glass slides via Cytospin  to form a single layer of 5 × 10PBL-1 cells were pelleted by centrifugation, fixed with 2.5% glutaraldehyde and 2% paraformaldehyde in 0.1 M phosphate buffer (pH 7.4) for 2 h at room temperature, post-fixed in 1% osmium tetroxide, and embedded in Epon. Ultrathin sections were stained with uranyl acetate and lead citrate, and observed under a transmission electron microscope  at 80 kV.54.RT-PCR to detect EBV latency–associated transcripts was performed as described previously5 cells/mL in RPMI 1640 medium containing 10% FBS in the presence or absence of 5 ng/mL IL-6 and/or applied drugs. After 48 h of culture, the XTT labelling mixture was added according to the manufacturer’s instructions. Colour development of formazan production was measured spectrophotometrically with Multiskan MS (LabSystems/Thermo Fisher Scientific) at 450 nm55. The cell proliferation ratio was calculated by the OD of a sample divided by the OD of the positive controls (IL-6-supplied PBL-1).The viability of PBL-1 cells was examined using a XTT (tetrazolium derivative) assay kit . Cells were cultured in 96-well plates in triplicate at a density of 2 × 1056. Briefly, 1 × 106 cells were lysed in 100 μL of M-PER lysis buffer containing Halt protease and phosphatase inhibitor cocktail . Cell lysates were subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis and transferred onto a polyvinylidene fluoride microporous membrane  using the NuPAGE system . The membranes were blocked with Block Ace and probed with primary antibodies followed by horse radish peroxidase-conjugated anti-mouse or anti-rabbit antibodies  with an immunoreaction enhancer solution . Blots were visualized by Super-Signal West Femto Chemiluminescent Substrate (Thermo Fisher Scientific) and images were captured with a C-Digit Blot Scanner .Protein extraction and immunoblotting were performed as described previouslyAnti-IL6R antibody, tocilizumab (Actemra), was purchased from Chugai Pharmaceutical, Tokyo, Japan, and used at 48 ng–100 μg/mL. To identify drugs that inhibited PBL-1 cell proliferation, PBL-1 cells were cultured in RPMI 1640 with 10% FBS, 5 ng/mL of IL-6, and drugs from the InhibitorSelect 96-well Protein Kinase Inhibitor Library I, II, III  in a 96-well plate at two final concentrations (10 nM and 1 μM). We also added 7 HDAC inhibitors (HDAC inhibitor Set II: Sigma Aldrich) and other antitumour drugs to PBL-1 cells with 5 ng/mL of IL-6 and cultured them for 48 h.50. XTT assay was performed after 48 hr of culture. A constant combination ratio experiment for the 8 point assay was carried out, as the ratio of (IC50 of drug 1)/(IC50 of drug 2) was constant. The Chou–Talalay Combination index (CI) was calculated according to a study by Chou et al. using CompuSyn Version 1.0 software (http://www.combosyn.com/)57. A CI value of <1, = 1, or >1 indicates synergy, additivity, and antagonism, respectively.PBL-1 cells were cultured in RPMI 1640 with 10% FBS, 5 ng/mL of IL-6 and two drugs  each added as a 2-fold dilution series from 6.4 μg/mL (tocilizumab), 32 nM (bortezomib and panobinostat) or 1.6 nM (everolimus) to determine the ICAll results were expressed as the mean ± SD of data obtained in triplicate, from at least three individual experiments. All statistical analyses were performed with Excel software , and GraphPad Prism Version 6.0 software . P values < 0.05 were considered statistically significant.The study protocol was approved by the Institutional Review Board, National Institute of Infectious Diseases  and Tokyo Metropolitan Komagome Hospital . Informed consent was obtained from the patient with PBL. All methods were performed in accordance with the relevant guidelines and regulations.Further methods are provided in the supplemental Materials and Methods.The datasets generated during and/or analysed during the current study are available from the corresponding author on reasonable request.Supplementary information


Thevetia peruviana (Pers.) K. Schum or Cascabela peruviana (L.) Lippold , native to Mexico and Central America, is a medicinal plant used traditionally to cure diseases like ulcers, scabies, hemorrhoids and dissolve tumors. The purpose of this study was to evaluate the cytotoxic, antiproliferative and apoptotic activity of methanolic extract of T. peruviana fruits on human cancer cell lines.T. peruviana methanolic extract was carried out on human breast, colorectal, prostate and lung cancer cell lines and non-tumorigenic control cells (fibroblast and Vero), using the MTT assay. For proliferation and motility, clonogenic and wound-healing assays were performed. Morphological alterations were monitored by trypan blue exclusion, as well as DNA fragmentation and AO/EB double staining was performed to evaluate apoptosis. The extract was separated using flash chromatography, and the resulting fractions were evaluated on colorectal cancer cells for their cytotoxic activity. The active fractions were further analyzed through mass spectrometry.The cytotoxic activity of T. peruviana methanolic extract exhibited cytotoxic activity on four human cancer cell lines: prostate, breast, colorectal and lung, with values of IC50 1.91 ± 0.76, 5.78 ± 2.12, 6.30 ± 4.45 and 12.04 ± 3.43 μg/mL, respectively. The extract caused a significant reduction of cell motility and colony formation on all evaluated cancer cell lines. In addition, morphological examination displayed cell size reduction, membrane blebbing and detachment of cells, compared to non-treated cancer cell lines. The T. peruviana extract induced apoptotic cell death, which was confirmed by DNA fragmentation and AO/EB double staining. Fractions 4 and 5 showed the most effective cytotoxic activity and their MS analysis revealed the presence of the secondary metabolites: thevetiaflavone and cardiac glycosides.The T. peruviana extract has potential as natural anti-cancer product with critical effects in the proliferation, motility, and adhesion of human breast and colorectal cancer cells, and apoptosis induction in human prostate and lung cancer cell lines, with minimal effects on non-tumorigenic cell lines.The online version of this article (doi:10.1186/s12906-017-1727-y) contains supplementary material, which is available to authorized users. Cancer is one of the most important causes of death worldwide. In 2012, 8.2 million cancer-related deaths were reported and the number of new cases is expected to rise by about 70% over the next two decades . TherefoCatharanthus, Nerium, Strophanthus, Apocynum and Thevetia , and then, DNA was extracted. The nucleic acid concentration and purity were measured using a NanoDrop® ND-2000 spectrophotometer (Thermo Scientific). Equal amounts of DNA (10 μg/well) were electrophoresed in 1% agarose gel. DNA fragments were visualized using an UV transilluminator . Three independent experiments were performed.DNA fragmentation was carried out as described previously . Cells w5 cells were seeded in a 6-well plate. Cells were treated with T. peruviana fruit extract at the corresponding IC50 concentration for 4 h. Then cells were subjected to AO/EB staining as described previously [For the AO/EB method, 5 × 10eviously . Briefly18 column equilibrated at 40 °C and a flow rate set at 0.3 mL/min. The mobile phase consisted of 15% CH3CN–H2O (0.1% formic acid) for 0.5 min, and then a linear gradient from 15% CH3CN to 100% CH3CN over 6 min, and 1 min holding 100% CH3CN before returning to the starting conditions. Samples were dissolved in MS grade methanol and filtered through a 0.2 μm Acrodisc (Waters) filter. Tentative metabolite identification was performed by comparison of HRMS data, UV maxima and fragmentation patterns (MS/MS data) with those contained in the Dictionary of Natural Products [Thevetia plant metabolites.HRESIMS data were collected and in positive and negative ionization modes using a Thermo QExactive Plus mass spectrometer (ThermoFisher) equipped an electrospray ionization (ESI) source and via an Acquity UPLC system (Waters Corp). The higher-energy collisional dissociation (HCD) cell used a normalized collision energy of 30 eV for all the compounds to obtain MS/MS data. The UPLC separation was performed using an Acquity BEH CProducts  referencp-value was analyzed in comparison to the untreated samples using Student’s t-Test or using one-way analysis of variance (ANOVA) followed by a Tukey test for comparison between different treatment groups. Differences were considered statistically significant at p < 0.05. Results were expressed as mean ± SEM of data obtained from tripled or quintuplet, independent experiments. Multivariate analysis was performed using the R 3.2.5 environment. Three biological replicates for each cell line were evaluated, and the variables used were the IC50, the clonogenic assay (CL), the membrane permeability (MP) and the wound-healing assay (WH). A standardized principal component analysis (PCA) was performed with “ade4” package [Statistical analysis was performed using GraphPad Prism 7 software. The  package , followe package .T. peruviana methanol extract on breast (HTB-22), colorectal (HTB-38), prostate (HTB-81), and lung (HTB-177) cancer cell lines, a MTT assay was performed. IC50 values indicate the concentration of the extract that inhibits the growth of 50% of the cell population. The criteria of cytotoxicity established by the U.S. National Cancer Institute (NCI) considers a crude extract as active, moderately active, or inactive, when the IC50 values are lower than 20 μg/mL, from 20 to 100 μg/mL, or higher than 100 μg/mL, respectively [T. peruviana methanol fruit extract induced strong cytotoxicity in all four cancer cell lines (<20 μg/mL), but prostate cancer cells showed the lowest IC50 after treatment with T. peruviana extract  on different types of cancer cells whereas the normal fibroblast cells were not affected.To determine whether the viability of the human cancer cells is affected during treatment with 25% Fig. . Interes50 value, the clonogenic assay, the wound healing assay, and membrane permeability. PCA was applied to the whole dataset and the first 3 principal components (PC’s) accounted for 97.3% of the observed variability. Assuming that directions with the greatest variance are the most biologically relevant, these leading PC’s were selected as those with the major structure or variance, as a de-noising step enhancing ICA results. ICA obtained 3 independent components  with negative kurtosis . The employment of these components, plotting IC1 and IC2, allowed the effective separation of samples into their original groups . The IC1 clearly separates tumor cells from normal cells, mainly due to the effect of the extract on cell motility  and the membrane permeability, while the IC2 separates samples mainly on the basis of the IC50 observed for each cell line. The anti-proliferative (clonogenic assay) response of the cell lines to the extract is mainly represented by the IC3, which can be observed in the Additional file T. peruviana extract were cell motility and membrane permeability and, less significantly, the IC50 and anti-proliferative activity.Multivariate analysis was performed on the six cell lines , using as variables to be evaluated: the ICT. peruviana fruit extract could induce DNA fragmentation and thus whether apoptosis occurred, human cancer cells exposed to T. peruviana treatment were assessed for DNA laddering and visualized by agarose gel electrophoresis  fluorescent staining was used to identify apoptosis-associated changes in the cell membrane. AO is a membrane-permeable dye that binds to the nucleic acids of viable cells. EB is an impermeable dye, but readily penetrates the membrane of nonviable cells and binds to DNA. When AO/EB are used simultaneously, viable cells fluoresce green and nonviable cells fluoresce red under fluorescence microscopy. Lung and prostate cells were labeled by AO/EB after 4 h of treatment with the T. peruviana extract, and dual staining was examined under a fluorescent microscope. Early and late-stage apoptotic cells, marked by crescent-shaped or granular yellow-green AO nuclear staining (as indicated by condensed or fragmented chromatin) were detected in the treated group (with T. peruviana extract) and the positive control (doxorubicin treated)  Fig. . Also, ded) Fig. . Thus, iT. peruviana fruit was subjected to flash chromatography and six fractions were obtained. Because prostate cancer cells showed the lowest IC50 to T. peruviana crude extract compared to lung, breast, and colorectal cancer cells, every single fraction was analyzed on the prostate cancer cell line by MTT assay, fractions four and five being the most significantly cytotoxic . The results showed that these fractions contained the polyketide thevetiaflavone and the cardiac glycosides: peruvosidic acid, peruvoside, thevefoline, solanoside, neriifoside and neriifolin.Methanol extract of ic Table . Those fT. peruviana belongs to the Apocynaceae family, a plant which is native to central and southern Mexico, as well as Central America. It is a medicinal plant used to treat different diseases, including cancer [T. peruviana on different types of human cancer cell lines.g cancer . The leag cancer , 28–30. T. peruviana fruit extract exhibited strong cytotoxicity against four cancer cell lines. Among the cell lines examined, lung cells showed higher IC50 value (12.04 μg/mL) than prostate (1.91 μg/mL), breast (5.78 μg/mL), and colorectal (6.30 μg/mL) cells. Such variation among the cell lines might be attributed in part to the fact that cancer cells possess differences in their genetic make up, morphology and doubling time, resulting in differential susceptibility to the same cytotoxic agent [T. peruviana fruit methanol extract has an antiproliferative potential.Our experiments in vitro showed that ic agent . Our resT. peruviana fruit extract significantly reduced the cell viability and the ability of cells to form colonies in four different human cancer cell lines. The clonogenic assay has been used to detect cells that have retained their capacity to produce a large number of progeny after radiation and chemotherapy treatments [T. peruviana fruit extract has potential anticancer activity, limiting proliferation of cancer cells after treatment. Notably, normal cells (vero and fibroblast) treated with T. peruviana extract did not show any observable effect in clonogenic assay.Cancer is a complex disease characterized by proliferation of highly resistant cells to death. An increased rate of cellular proliferation is frequent, due to the fact that most cancer cells divide more often than normal cells. The goal of targeting cell proliferation is to arrest the cell cycle or induce cancer cell death using cytotoxic compounds. We show here, that eatments . It alsoeatments . Hence, T. peruviana fruit extract inhibited and delayed the cell migration of cancer cells, but not among normal cells. These results open the door to further studies that could confirm if the cytotoxic activity of T. peruviana fruit extract alters the regulation of the actin cytoskeleton, induces morphological changes and leads to detachment of cells, culminating in cell death. Moreover, motility and membrane permeability features were mainly affected by T. peruviana fruit extract, according with the multivariate analysis performed on the six cell lines and the four variables evaluated, suggesting a potential antimetastatic activity in the T. peruviana fruit extract.Migration is a critical step in initial progression of cancer that facilitates metastasis. Wound and healing assay is a classic and common method used for discovery and validation of molecules that affect cell migration , 34 and Cerbera manghas, a plant belonging to the Apocynaceae family, contains a cardiac glycoside (neriifolin), which induced DNA fragmentation on hepatocellular carcinoma 48 h after treatment [T. peruviana fruit showed death induction on cancer cell lines through early apoptosis mechanisms (AO/EB fluorescent staining) 4 h after treatment and late-stage apoptosis (DNA laddering assay) after 24 h of treatment. This result indicates that T. peruviana could have a higher level of apoptotic activity than other members of the Apocynaceae family. In addition, it will be important to determine whether the apoptotic activity is located in the compounds present in the flesh or seeds.One crucial and desirable mechanism by which chemotherapeutics destroy tumor cells is by inducing apoptosis. Cells undergoing apoptosis show morphological and biochemical modifications including chromatin segregation, nuclear condensation, DNA fragmentation, partition of the membrane, and vesicles formation , 37. Thereatment . Here, cT. peruviana fruit methanol extract indicated that one flavonoid and cardiac glycosides are secondary metabolites present in the fruit plant. Flavonoids, such as curcumin, quercentin and genistein, are known to have cell line-specific anti-proliferative and apoptosis inducing activity [T. peruviana on human cancer cell lines. Recent investigations of seeds from T. peruviana resulted in the isolation of cardiac glycosides that had inhibitory effects against human gastric and pancreatic cancer cell lines [T. peruviana due to the fact they can heal heart pathologies [T. peruviana fruit methanolic extract, mainly due to the effect of the extract on the motility and membrane permeability on human prostate, breast, colorectal and lung cancer cell lines. The cytotoxic activity of crude extract from T. peruviana fruit could be attributed to their phytochemical components such as thevetin A, thevetin B, peruvoside, thevenerin, and cerberin, which are toxins [T. peruviana fruit is currently being undertaken to investigate their cytotoxic, molecular and genetic action mechanisms, which could provide meaningful perspectives for biomedical and biotechnological research.Mass spectrometry analysis of active fractions from activity –42. It hactivity –46. Furtll lines . Cardiachologies , but arehologies  and hepahologies . Thevetihologies . One hune toxins , and alsT. peruviana fruit inhibits cell proliferation, has a time-dependent cytotoxic activity and induces apoptosis of human cancer cell lines, but has minimal or less pronounced effects on normal cells. The fruit extract displayed anticancer properties mainly through mechanisms that included membrane permeability, motility and DNA fragmentation. Maximum cytotoxic activity was observed in a fraction that contained one flavonoid and cardiac glycosides. Chemical analyses of the active fractions are currently in progress to perform a better evaluation of their biological significance. Additionally, further “in vivo” research is essential to show the full potential for the use of T. peruviana fruit extract in cancer therapy. In conclusion, these findings shows the importance of T. peruviana fruit as a source of bioactive compounds with anticancer potential.The current study presents evidence that the methanol extract of Additional file 1: Figure S1.Clonogenic assay of four human cancer cell lines treated with doxorubicin. Image showing colonies produced by human cancer lines and normal cell lines, following plating of 100 cells and 10–14 days incubation. Cells were treated with doxorubicin at 10 μg/mL. (PDF 1719 kb)Additional file 2: Figure S2.T. peruviana extract. Representative photographs of wounded cancer cells monolayer after 24 h of treatment with methanol T. peruviana fruit extract at IC50 value corresponding to each cell line. Vero and fibroblast cells were used as normal cell lines. A typical result from three independent experiments is shown. (PDF 2.74 mb)Wound and healing closure activity of four human cancer cell lines treated with methanol Additional file 3: Figure S3.Wound and healing closure activity of four human cancer cell lines treated with doxorubicin. Representative photographs of wounded cancer cells monolayer after 24 h of treatment with doxorubicin at 10 µg/mL. The result from three independent experiments is shown. (PDF 2.62 mb)Additional file 4: Figure S4.Morphological changes on four human cancer cell lines during treatment with doxorubicin. Human cancer cells were treated with doxorubicin at 10 µg/mL and monitored over a 24 h period. (PDF 1.88 mb)Additional file 5:Figure S5. Classification of normal and cancer cell lines exposed to T. peruviana fruit methanolic extract according to independent component analysis (ICA). The distribution of the cell lines  and variables  is shown in the space spanned by the independent components 1 and 3. The clear unsupervised discrimination among the six cell lines reflects the greater effect of T. peruviana extract on tumor cell lines , while normal cells are affected less . The independent component 1 is clearly separating cancer cells from normal cells, mainly due to the effect of the extract on the motility (WH) and membrane permeability (MP), while the independent component 3 is separating samples mainly by the anti-proliferative  observed for each cell line. (PDF 1121 kb)