Human Burkitt Lymphoma Research Articles

Enhanced electrochemiluminescence ratiometric cytosensing based on surface plasmon resonance of Au nanoparticles and nanosucculent films

Ramos cells are human Burkitt's lymphoma cells, which are a kind of cancer cells to facilitate the monitoring of the relevant biological processes of cancers. Sensitive and accurate detection of Ramos cells using emerging ratiometric ECL biosensing technology shows increasing importance, however, the target analytes of current ratiometric ECL biosensors are mainly limited to DNA/RNA or proteins. In this study, we proposed a dual-potential ratiometric sensing strategy for the electrochemiluminescence detection of Ramos cells based on two types of electrochemiluminescence (ECL)-responding molecular. Au nanosucculent films (AuNFs) were electrodeposited on the fluorine doped tin oxide (FTO) electrode to increase the effective area of the electrode for more efficient assembly of DNA and effectively improving the conductivity of the sensing interfaces. In the presence of Ramos cells, aptamers capped with Au@luminol would conjugate with Ramos cells and then remove from AuNFs, accompanying the decrease of ECL signal from Au@luminol. Then, Au–DNA was captured and alternately hybridized with DNA-modified CdS nanocrystals (NCs) on the surface of AuNFs with the formation of a super reticulate structure. The reticulate structure not only raised another identified ECL signal from CdS NCs but also greatly promoted its ECL intensity from the surface plasmon resonance originating from Au NPs. The value of log (ECLCdS/ECLluminol) and the logarithm of the number of cells exhibit considerable linear relation ranging from 80 to 8 × 105 cells mL−1 with a low detection limit of 20 cells mL−1 (S/N = 3). The selectivity and specificity of this dual-potential ECL sensor showed good performance and indicated considerable promise in avoiding false-positive results in detection.

Effect of Autophagy to Drug Resistance of B-cell Lymphoma Cells

To investigate the effect of autophagy on the drug resistance of different human lymphoma cells. Human Burkitt's lymphoma cell Daudi, human B lymphoma cell SUDHL-4, and human mantle cell lymphoma cell JeKo-1 were taken as the research subjects. The expression of Atg5 was inhibited by the treatments of autophagy inhibitors or stable interference via lentivirus infection. The autophagy activity of B lymphoma cell was changed, and the changes of lymphoma cells to the drug resistance of ADR and VCR was observed. JeKo-1 cells showed the strongest resistance to ADR and VCR, followed by SUDHL-4, and Daudi cells showed the weakest resistance to ADR and VCR. At the same time, JeKo-1 cells showed the strongest autophagy activity, followed by SUDHL-4, and Daudi cells showed the weakest autophagy activity. After the treatments of autophagy inhibitors or stable Atg5 interference, the resistance of lymphoma cells to ADR and VCR was significantly weakened, and there was the positive correlation at the drug resistance and the autophagy activity of B lymphoma cell. The higher autophagy activity in lymphoma cells, the lower chemotherapy resistance of the lymphoma cells after autophagy was inhibited.

Nanoencapsulated rituximab mediates superior cellular immunity against metastatic B-cell lymphoma in a complement competent humanized mouse model

BackgroundDespite the numerous applications of monoclonal antibodies (mAbs) in cancer therapeutics, animal models available to test the therapeutic efficacy of new mAbs are limited. NOD.Cg-Prkdcscid Il2rgtm1Wjl/SzJ (NSG) mice are one...

MYC regulates ribosome biogenesis and mitochondrial gene expression programs through its interaction with host cell factor-1.

The oncoprotein transcription factor MYC is a major driver of malignancy and a highly validated but challenging target for the development of anticancer therapies. Novel strategies to inhibit MYC may come from understanding the co-factors it uses to drive pro-tumorigenic gene expression programs, providing their role in MYC activity is understood. Here we interrogate how one MYC co-factor, host cell factor (HCF)-1, contributes to MYC activity in a human Burkitt lymphoma setting. We identify genes connected to mitochondrial function and ribosome biogenesis as direct MYC/HCF-1 targets and demonstrate how modulation of the MYC-HCF-1 interaction influences cell growth, metabolite profiles, global gene expression patterns, and tumor growth in vivo. This work defines HCF-1 as a critical MYC co-factor, places the MYC-HCF-1 interaction in biological context, and highlights HCF-1 as a focal point for development of novel anti-MYC therapies.

The Antimicrobial Peptide, Nisin, Synergistically Enhances the Cytotoxic and Apoptotic Effects of Rituximab Treatment on Human Burkitt's Lymphoma Cell Lines.

Non-Hodgkin's lymphomas comprise the most common hematological cancers worldwide and consist of a heterogenous group of malignancies affecting the lymphoid system. Treatment of non-Hodgkin's lymphoma has been significantly enhanced with the addition of Rituximab to the standard chemotherapy regimen. However, even with the advancement of treatment patients continue to relapse and develop resistance to Rituximab, rendering subsequent treatments unsuccessful. The use of novel drugs with unique antitumor mechanisms has gained considerable attention. In this study, we explored the in vitro anti-cancer effects of the combined therapy of Rituximab and Nisin on human Burkitt's lymphoma cells. The human Burkitt's lymphoma cells lines, Raji and Daudi, were treated with Nisin, Rituximab, or a combination of the two agents at various concentrations. Cytotoxicity following treatment was determined using cell viability assay. The degree of apoptosis was verified via flow cytometric analysis using FITC annexin V/PI staining. Our findings show that the combined treatment of Rituximab and Nisin results in a more significant reduction in the survival of Raji and Daudi Burkitt's lymphoma cells, compared to Nisin or Rituximab treatment alone. Additionally, our results indicate that Nisin can induce a significant degree of apoptosis in the Burkitt's lymphoma cells compared to the negative controls. However, the addition of Nisin to the Rituximab treatment synergistically enhances the apoptotic antitumor effect. This study demonstrates the synergistic antitumor effect of Nisin treatment in vitro to enhance tumor cell apoptosis and the potential value of Nisin as an adjunct therapy in the treatment of lymphoma.

Novel cytokine–antibody fusion protein, N-820, to enhance the functions of ex vivo expanded natural killer cells against Burkitt lymphoma

BackgroundThe prognosis of patients with relapsed or progressive B cell (CD20+) non-Hodgkin’s lymphoma (B-NHL), including Burkitt lymphoma (BL), is dismal due to chemoradiotherapy resistance. Novel therapeutic strategies are urgently needed....

Inhibitory effect of simiao qingwen baidu decoction on epstein-barr virus EA, VCA expression and DNA replication in vitro

This article aims to investigate the role of Simiao Qingwen Baidu Decoction (traditional Chinese medicine) in Epstein-Barr virus (EBV)-induced infectious mononucleosis. Sprague Dawley rats were given Simiao Qingwen Baidu Decoction by gavage, and the medicated serum was collected. EBV-latent infected human Burkitt lymphomas Raji and EBV-transformed marmosets B lymphoblast cell B95−8 were treated with medicated serum. CCK8 assay and flow cytometry were performed to detect cell proliferation and apoptosis. Indirect immunofluorescence assay was performed to analyze EA or VCA positive expression. The copy-number of EBV-DNA and the gene expression were detected by quantitative PCR or quantitative real-time PCR. We found that the medicated serum inhibited proliferation of Raji and B95−8 cells, especially 10 %-medicated serum. The 10 %-medicated serum significantly suppressed EA expression in Raji cells and VCA expression in B95−8 cells. The expression of BZLF1, BRLF1, BMLF1 and EBNA-1 in Raji cells was significantly inhibited by 10 %-medicated serum. 10 %-medicated serum caused a decrease in the copy-number of EBV-DNA in Raji cells. In conclusion, our data imply that Simiao Qingwen Baidu Decoction represses the expression of EA and VCA, and EBV-DNA replication. Thus, our work suggests that Simiao Qingwen Baidu Decoction may play a vital role in anti-EBV.

Abstract 5655: Efficacy of antibodies targeting TfR1 in xenograft mouse models of AIDS-related non-Hodgkin lymphoma

Abstract Infection with the human immunodeficiency virus (HIV) and the development of acquired immunodeficiency syndrome (AIDS) increases the risk of developing B-cell lymphomas. AIDS-related B-cell non-Hodgkin lymphoma (AIDS-NHL) remains a significant clinical problem in the era of effective combination anti-retroviral therapy. In fact, AIDS-NHL is currently the most common AIDS-related malignancy in developed countries, where NHL accounts for 23-30% of all AIDS-related deaths. Transferrin receptor 1 (TfR1), also known as CD71, is a type II transmembrane homodimeric protein involved in the cellular uptake of iron and the regulation of cell growth. It is expressed on normal B cells at low levels and is upregulated upon activation. HIV infection leads to the chronic activation of B cells, which results in high expression of TfR1 on these cells, B-cell dysfunction, and ultimately the development of AIDS-NHL. Importantly, TfR1 expression is correlated with stage and prognosis of NHL including AIDS-NHL. Thus, it is a meaningful target for antibody-based NHL therapy. We previously developed a mouse/human chimeric IgG3 specific for human TfR1 (ch128.1/IgG3) and have shown that this antibody administered intraperitoneally exhibits antitumor activity in an in vivo model of AIDS-NHL where NOD-SCID mice were challenged intraperitoneally with 2F7 human Burkitt lymphoma (BL) cells that are positive for the Epstein-Barr Virus (EBV). More recently, we have developed a mouse/human chimeric IgG1 version of ch128.1 that shows significant antitumor activity in various disseminated mouse models of human multiple myeloma (MM), another B-cell malignancy. In the present studies, we sought to further explore in two novel mouse models of human AIDS-NHL the utility of targeting the TfR1 with ch128.1/IgG1 and its recently developed humanized version (hu128.1). To accomplish this goal, we used the 2F7 cell line variant 2F7-BR44, which forms metastases in the brain of mice upon intravenous (i.v.) injection. We also used human JB cells, which are of particular relevance since they are human BL cells that are EBV negative. We found that systemic (i.v.) treatment with ch128.1/IgG1 or hu128.1 of SCID-Beige mice challenged locally (subcutaneously) with 2F7-BR44 or JB tumor cells results in significant antitumor activity against different stages of the disease. Treatment of mice challenged systemically (i.v.) with 2F7-BR44 or JB tumor cells also showed antitumor activity, including long-term survival in some cases. Taken together, our results suggest that targeting TfR1 with antibodies such as ch128.1/IgG1 and hu128.1 has potential as an effective therapy for B-cell malignancies, including MM and AIDS-NHL. Citation Format: Tracy R. Daniels-Wells, Pierre V. Candelaria, Emiko Kranz, Jing Wen, Lan Weng, Masakazu Kamata, Juan Carlos Almagro, Otoniel Martinez-Maza, Manuel L. Penichet. Efficacy of antibodies targeting TfR1 in xenograft mouse models of AIDS-related non-Hodgkin lymphoma [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 5655.

Establishment of an Anti-CD20 Monoclonal Antibody (C20Mab-60) for Immunohistochemical Analyses.

Sensitive and specific monoclonal antibodies (mAbs) are needed for detecting CD20. This antigen, one of several B lymphocyte antigens, is a target for every application used in the diagnosis of B cell lymphoma. Many anti-CD20 mAbs have been established, although applications of these antibodies are limited. This study aims to establish sensitive and specific anti-CD20 mAbs suitable for broad application, such as flow cytometry, Western blotting, and immunohistochemical analyses. Using the Cell-Based Immunization and Screening (CBIS) method, all procedures were performed by utilizing CD20-stable transfectants, and a clone, C20Mab-60 (IgG2a, kappa), was developed. In flow cytometry, C20Mab-60 detected overexpression of CD20 in LN229 cell and endogenous CD20 in BALL-1 (a human B cell leukemia cell line) but did not react with CD20-knockout BALL-1 (BINDS-24), indicating specificity for CD20. In Western blotting, C20Mab-60 detected CD20-overexpressing Chinese hamster ovary-K1, BALL-1, and Raji (a human Burkitt's lymphoma cell line) displaying both sensitivity and specificity. Furthermore, B cell but not T cell lymphomas were strongly stained with C20Mab-60 in immunohistochemical analyses. C20Mab-60, which was developed by CBIS method, is shown to be useful for the detection of cells expressing CD20 in lymphoma tissues by flow cytometry, Western blotting, and immunohistochemical analyses.

Characterization of resistance to a recombinant hexameric Fas-ligand (APO010) in human cancer cell lines

Multiple myeloma remains a hard-to-treat cancer as all patients eventually progress because of drug resistance. Thus, there is a need for novel and non-cross-resistant treatment options, and we aimed to address this issue by introducing a new immuno-oncology drug (APO010) in multiple myeloma treatment. APO010 is a hexameric Fas-ligand that mimics cytotoxic T-lymphocyte signaling through the Fas-receptor to induce apoptosis. APO010 is currently in clinical trials with multiple myeloma patients. Thus, an understanding of the mechanisms contributing to resistance to APO010 will be essential for future clinical studies with APO010, and it might be possible to develop strategies to circumvent this resistance. We developed APO010-resistant variants of human multiple myeloma cell lines (LP1, MOLP-8, and KMS-12-BM) and a human Burkitt's lymphoma cell line (Raji) by exposing the cells to gradually increasing concentrations of APO010 over a period of 6-12 months. The resistant cell lines were characterized on the basis of immunocytochemistry, Fas-receptor protein expression, mRNA expression analysis, and pathway analysis. APO010-resistant cell lines exhibited a 4- to 520-fold increase in resistance to APO010 and still remained sensitive to other chemotherapeutics. Downregulation of the Fas-receptor protein expression was observed in all resistant cell lines. mRNA expression analysis of the resistant versus parental cell lines confirmed a significant alteration in FAS expression between sensitive and resistant cell lines (p = 0.03), while pathway analysis revealed alterations in mRNA signaling pathways of Fas. On the basis of the pre-clinical data obtained, it can be concluded that downregulation of Fas-receptor can mediate resistance to APO010.

EBNA2-deleted Epstein-Barr virus (EBV) isolate, P3HR1, causes Hodgkin-like lymphomas and diffuse large B cell lymphomas with type II and Wp-restricted latency types in humanized mice.

EBV transforms B cells in vitro and causes human B-cell lymphomas including classical Hodgkin lymphoma (CHL), Burkitt lymphoma (BL) and diffuse large B-cell lymphoma (DLBCL). The EBV latency protein, EBNA2, transcriptionally activates the promoters of all latent viral protein-coding genes expressed in type III EBV latency and is essential for EBV’s ability to transform B cells in vitro. However, EBNA2 is not expressed in EBV-infected CHLs and BLs in humans. EBV-positive CHLs have type II latency and are largely driven by the EBV LMP1/LMP2A proteins, while EBV-positive BLs, which usually have type I latency are largely driven by c-Myc translocations, and only express the EBNA1 protein and viral non-coding RNAs. Approximately 15% of human BLs contain naturally occurring EBNA2-deleted viruses that support a form of viral latency known as Wp-restricted (expressing the EBNA-LP, EBNA3A/3B/3C, EBNA1 and BHRF1 proteins), but whether Wp-restricted latency and/or EBNA2-deleted EBV can induce lymphomas in humanized mice, or in the absence of c-Myc translocations, is unknown. Here we show that a naturally occurring EBNA2-deleted EBV strain (P3HR1) isolated from a human BL induces EBV-positive B-cell lymphomas in a subset of infected cord blood-humanized (CBH) mice. Furthermore, we find that P3HR1-infected lymphoma cells support two different viral latency types and phenotypes that are mutually exclusive: 1) Large (often multinucleated), CD30-positive, CD45-negative cells reminiscent of the Reed-Sternberg (RS) cells in CHL that express high levels of LMP1 but not EBNA-LP (consistent with type II viral latency); and 2) smaller monomorphic CD30-negative DLBCL-like cells that express EBNA-LP and EBNA3A but not LMP1 (consistent with Wp-restricted latency). These results reveal that EBNA2 is not absolutely required for EBV to form tumors in CBH mice and suggest that P3HR1 virus can be used to model EBV positive lymphomas with both Wp-restricted and type II latency in vivo.

BI6727, a polo-like kinase 1 inhibitor with promising efficacy on Burkitt lymphoma cells

ObjectiveBI6727, an ATP-competitive PLK1 inhibitor, has been shown to cause cell death in multi-tumors. This study aimed to investigate the anti-tumor effect and potential molecular mechanism of BI6727 in human Burkitt lymphoma (BL) cell lines.MethodsWe assessed polo-like kinase 1 (PLK1) expression in BL patient tissues and cells, also investigated the cytotoxic effect using CCK8 assay and flow cytometry. In addition, western blotting and real-time polymerase chain reaction (RT-PCR) assays were used to explore the molecular mechanisms of BI6727 in human BL cell lines.ResultsPLK1 was overexpressed in BL cells compared with normal cells. The PLK1 inhibitor BI6727 reduced activated PLK1 expression and caused mitotic arrest in BL cells. Additionally, BI6727 suppressed cellular proliferation and induced apoptosis in BL cell lines. BI6727 treatment also decreased C-MYC protein and mRNA expression, blocked the PI3K/AKT/mTOR signaling pathway, and stabilized the FBXW7 protein.ConclusionsOur findings explained a potential molecular mechanism of BI6727 in BL cells and suggested that BI6727 might be a new therapeutic agent for BL in the future.

BCL-W is dispensable for the sustained survival of select Burkitt lymphoma and diffuse large B-cell lymphoma cell lines

AbstractDysregulated expression of BCL-2 family proteins allows cancer cells to escape apoptosis. To counter this, BH3-mimetic drugs that target and inhibit select BCL-2 prosurvival proteins to induce apoptosis have been developed for cancer therapy. Venetoclax, which targets BCL-2, has been effective as therapy for patients with chronic lymphocytic leukemia, and MCL-1–targeting BH3-mimetic drugs have been extensively evaluated in preclinical studies for a range of blood cancers. Recently, BCL-W, a relatively understudied prosurvival member of the BCL-2 protein family, has been reported to be abnormally upregulated in Burkitt lymphoma (BL), diffuse large B-cell lymphoma (DLBCL), and Hodgkin lymphoma patient samples. Therefore, to determine if BCL-W would be a promising therapeutic target for B-cell lymphomas, we have examined the role of BCL-W in the sustained growth of human BL- and DLBCL-derived cell lines. We found that CRISPR/CAS9-mediated loss or short hairpin RNA-mediated knockdown of BCL-W expression in selected BL and DLBCL cell lines did not lead to spontaneous apoptosis and had no effect on their sensitivity to a range of BH3-mimetic drugs targeting other BCL-2 prosurvival proteins. Our results suggest that BCL-W is not universally required for the sustained growth and survival of human BL and DLBCL cell lines. Thus, targeting BCL-W in this subset of B-cell lymphomas may not be of broad therapeutic benefit.

Eμ and 3′RR transcriptional enhancers of the IgH locus cooperate to promote c-myc–induced mature B-cell lymphomas

AbstractNumerous B-cell lymphomas feature translocations linking oncogenes to different locations in the immunoglobulin heavy chain (IgH) locus. During Burkitt lymphoma (BL), IgH breakpoints for c-myc translocation stand either close to JH segments or within switch regions. Transcription, accessibility, and remodeling of the IgH locus are under the control of the 2 potent cis-acting enhancer elements: Eμ and the 3′ regulatory region (3′RR). To ensure their respective contributions to oncogene deregulation in the context of the endogenous IgH locus, we studied transgenic mice harboring a knock-in of c-myc in various positions of the IgH locus (3′ to JH segments, 5′ to Cμ with Eμ deletion and Cα). The observed spectrum of tumors, kinetics of emergence, and transcriptome analysis provide strong evidence that both Eμ and 3′RR deregulate c-myc and cooperate together to promote B-cell lymphomagenesis. Transgenics mimicking endemic BL (with c-myc placed 3′ to JH segments) exhibited the highest rate of B-cell lymphoma emergence, the highest Ki67 index of proliferation, and the highest transcriptomic similarities to human BL. The 3′RR enhancer alone deregulated c-myc and initiated the development of BL-like lymphomas, suggesting that its targeting would be of therapeutic interest to reduce c-myc oncogenicity in vivo.

Histone deacetylase inhibition enhances the therapeutic effects of methotrexate on primary central nervous system lymphoma.

BackgroundPolyglutamylation is a reversible protein modification that commonly occurs in tumor cells. Methotrexate (MTX) in tumor cells is polyglutamylated and strongly binds to dihydrofolate reductase (DHFR) without competitive inhibition by leucovorin. Therefore, tumor cells with high polyglutamylation levels are supposed to be selectively killed, whereas normal cells with lower polyglutamylation are rescued by leucovorin. This study investigated the combined effects of MTX plus histone deacetylase inhibitors (HDACIs), which upregulate MTX polyglutamylation, in primary central nervous system lymphoma (PCNSL).MethodsWe evaluated cell viability after MTX treatment and leucovorin rescue and compared the expression of folylpolyglutamate synthetase (FPGS), γ-glutamyl hydrolase (GGH), and DHFR in 2 human PCNSL-derived cell lines (HKBML and TK) and a human Burkitt lymphoma cell line (TL-1). Combination treatments were created using 4 HDACIs: panobinostat, vorinostat, sodium butyrate, and valproic acid. The expression of DHFR was examined as well as ratios of FPGS/GGH expression. The combined effects of MTX plus HDACIs were evaluated using a cell viability assay, mass spectroscopy imaging, and subcutaneous and intracranial xenograft models.ResultsHDACIs upregulated the ratio of FPGS/GGH expression resulting in increased polyglutamylation of MTX, but also downregulated expression of the target molecule of MTX: DHFR. The combination of MTX and vorinostat decreased cell viability in vitro (P < .05) and tumor volumes in a subcutaneous model (P < .0001), and prolonged survival in an intracranial model (P < .01), relative to controls.ConclusionHDACIs enhanced the therapeutic effect of MTX through increased polyglutamylation of MTX and concomitant downregulation of DHFR expression.

The effect of Yarrowia lipolytical-asparaginase on apoptosis induction and inhibition of growth in Burkitt's lymphoma Raji and acute lymphoblastic leukemia MOLT-4 cells

l-Asparaginase (l-asparagine amidohydrolase; E.C.3.5.1.1) as an anticancer agent is used to treat acute lymphocytic leukemia (ALL), Human Burkitt's lymphoma and non-Hodgkin's lymphoma. The commercial asparaginases are obtained from bacteria Erwinia chrysanthemi and Escherichia coli now which had many side effects. In this study, the effects of a novel l-asparaginase from yeast Yarrowia lipolytica was investigated on human ALL and Burkitt's lymphoma cell lines. The l-asparaginase causes metabolic stress, cytotoxicity, and apoptosis due to the arrest of the G0 cell cycle, the activation of caspase-3 and the modulation of mitochondrial membrane integrity. The RT-PCR analysis showed a significant increase in the pro-apoptosis genes such as Bax, Caspase-3, Caspase-8, Caspase-9 and p53 (P < 0.05) while the anti-apoptotic marker Bcl-2 was significantly decreased (P < 0.05). Furthermore, Y. lipolytical-asparaginase causes autophagy and increased ROS. The l-asparaginase has cytotoxic and anticancer effects higher than commercial asparaginase. In conclusion, Y. lipolytical-asparaginase shows interesting anticancer activity and it can be introduced as a new eukaryotic and therapeutic agent and strategy for ALL and Burkitt's lymphoma treatment after the in vivo and clinical experiments.

GCN5 HAT inhibition reduces human Burkitt lymphoma cell survival through reduction of MYC target gene expression and impeding BCR signaling pathways.

GCN5, the catalytic subunit in the acetyltransferase modules of SAGA and ATAC, functions as a coactivator of gene transcription. The SAGA complex is recruited to chromatin by transcription factors such as MYC and E2F1 to facilitate acetylation of histones, especially H3 at lysine 9 (H3K9). Burkitt lymphoma is an aggressive subtype of Non-Hodgkin lymphoma driven by the overexpression of MYC. Comparison of GCN5 expression in normal human B cells versus human Burkitt Lymphoma cell lines indicates overexpression of GCN5 in lymphoma. Treatment of Burkitt lymphoma cell lines with a specific inhibitor indicates that decreased GCN5 HAT activity reduces viability and proliferation of these cells. Inhibition of GCN5 HAT activity also induces apoptosis in lymphoma cells. Expression of MYC target genes as well as genes associated with B cell receptor signaling are significantly downregulated upon inhibition of GCN5 enzymatic activity. This downregulation leads to diminished PI3K signaling, a critical pathway in lymphomagenesis. Our data indicate that inhibition of GCN5 HAT activity reduces the tumorigenic properties of human Burkitt lymphoma cells by attenuating BCR signaling and that GCN5 may be a viable target for lymphoma drug therapy.

Effect of Baicalin Derivative 02-036 on Burkitt Lymphoma Cell Line CA46 and Its Related Mechanisms

To investigate the effect of baicalin derivative 02-036 on proliferation and apoptosis human Burkitt lymphoma cell line CA46 and its related mechanisms. The MTT assay and cell colony formation assay were used to measure the growth inhibition of CA46 cells after 02-036 treatment. The flow cytometry with AnnexinV-FITC/PI double staining was employed to detect the apoptosis induction effect of 02-036 on CA46 cells. Cell cycle distribution of CA46 cells was estimeted by using DNA ploid analysis. Western blot was used to determine the changes of apoptosis-related proteins, including C-MYC, BCL-2, Procaspase-9, Procaspase-3, PARP and Cleaved-PARP. Baicalin derivative 02-036 obviously inhibited the proliferation of CA46 cells, with dose- and time-dependent manner (r=0.963, r=0.992). The averaged IC50 value of CA46 cells was (6.04±0.11) μmol/L after 48-hour treatment. Low concentration of 02-036 could significantly inhibit the colony formation of CA46 cells. Flow cytometry analysis confirmed that 02-036 could effectively induce CA46 cell apoptosis. The apoptosis rate correlated with drug concentrations (r=0.959). Also, DNA ploid analysis showed that the cell cycle of CA46 was arrested in the S phase. The expression levels of BCL-2, Pro-caspase-9, Pro-caspase-3, PARP and C-MYC proteins decreased with a 02-036-dose dependent manner (r values were -0.990, -0.939, -0.971 and -0.967, respectively). In contrast, the expression level of cleaved-PARP increased with the same manner (r=0.920). Baicalin derivative 02-036 can effectively inhibit the proliferation and induce apoptosis of CA46 cells, and its related mechanisms may be correlated with the down-regulation of apoptosis-related molecule expression levels, such as BCL-2, Pro-caspase-9, Pro-caspase-3, PARP and C-MYC.

FOXO1 Confers Maintenance of the Dark Zone Proliferation and Survival Program and Can Be Pharmacologically Targeted in Burkitt Lymphoma.

The FOXO1 transcription factor plays a central role in the proliferation and survival of B cells at several stages of differentiation. B cell malignancies, with exception of classical Hodgkin lymphoma, maintain expression of FOXO1 at levels characteristic for their non-malignant counterparts. Extensive expression profiling had revealed that Burkitt lymphoma (BL) show many characteristics of the dark zone (DZ) germinal center (GC) B cell program. Here we show that FOXO1 knockdown inhibits proliferation of human BL cell lines. The anti-proliferative effect of the FOXO1 knockdown is associated with the repression of the DZ B cell program including expression of MYB, CCND3, RAG2, BACH2, and CXCR4. In addition, the induction of signaling pathways of the light zone (LZ) program like NF-κB and PI3K-AKT was observed. Using a rescue experiment we identified downregulation of the proto-oncogene MYB as a critical factor contributing to the antiproliferative effect of FOXO1 knockdown. In an attempt to estimate the feasibility of pharmacological FOXO1 repression, we found that the small molecular weight FOXO1 inhibitor AS1842856 induces cell death and growth arrest in BL cell lines at low concentrations. Interestingly, we found that overactivation of FOXO1 also induces growth inhibition in BL cell lines, indicating the importance of a tight regulation of FOXO1 activity in BL.

Regulatory T Cells in an Endogenous Mouse Lymphoma Recognize Specific Antigen Peptides and Contribute to Immune Escape.

Foxp3+ regulatory T cells (Tregs) sustain immune homeostasis and may contribute to immune escape in malignant disease. As a prerequisite for developing immunologic approaches in cancer therapy, it is necessary to understand the ontogeny and the antigenic specificities of tumor-infiltrating Tregs. We addressed this question by using a λ-MYC transgenic mouse model of endogenously arising B-cell lymphoma, which mirrors key features of human Burkitt lymphoma. We show that Foxp3+ Tregs suppress antitumor responses in endogenous lymphoma. Ablation of Foxp3+ Tregs significantly delayed tumor development. The ratio of Treg to effector T cells was elevated in growing tumors, which could be ascribed to differential proliferation. The Tregs detected were mainly natural Tregs that apparently recognized self-antigens. We identified MHC class II-restricted nonmutated self-epitopes, which were more prevalent in lymphoma than in normal B cells and could be recognized by Tregs. These epitopes were derived from proteins that are associated with cellular processes related to malignancy and may be overexpressed in the tumor.