Burkitt's Lymphoma Raji Cells Research Articles

Reversal Effect of NVP-BEZ235 on Doxorubicin-Resistance in Burkitt Lymphoma RAJI Cell Line

To study the reversal effect of NVP-BEZ235 on doxorubicin resistance in Burkitt lymphoma RAJI cell line. The doxorubicin-resistant cell line was induced by treating RAJI cells with a concentration gradient of doxorubicin. The levels of Pgp, p-AKT, and p-mTOR in cells were detected by Western blot. Cell viability was detected by MTT assay. IC50 was computed by SPSS. The doxorubicin-resistant Burkitt lymphoma cell line, RAJI/DOX, was established successfully. The expression of Pgp and the phosphorylation levels of AKT and mTOR in RAJI/DOX cell line were both higher than those in RAJI cell line. NVP-BEZ235 downregulated the phosphorylation levels of AKT and mTOR in RAJI/DOX cell line. NVP-BEZ235 inhibited the proliferation of RAJI/DOX cell line, and the effect was obvious when it was cooperated with doxorubicin. The constitutive activation of PI3K/AKT/mTOR pathway of RAJI/DOX cell line was more serious than RAJI cell line. NVP-BEZ235 reversed doxorubicin resistance of RAJI/DOX cell line by inhibiting the PI3K/AKT/mTOR signal pathway.

The novel high-affinity humanized antibody IMM40H targets CD70, eliminates tumors via Fc-mediated effector functions, and interrupts CD70/CD27 signaling.

A significant level of CD70 can be detected in various types of tumor tissues and CD27 is expressed on Treg cells, but CD70 expression is low in normal tissues. The interaction between CD70 and CD27 can stimulate the proliferation and survival of cancer cells and increase the level of soluble CD27, which is associated with poor prognosis in patients with lymphoma and certain solid tumors. Thus, it is a promising therapeutic target for the treatment of many major CD70+ cancer indications, including CD70+ lymphoma, RCC, NSCLC, HNSCC and OC. IMM40H was obtained through hybridoma screening and antibody humanization techniques. IMM40H was evaluated for its binding, blocking, Fc-dependent effector functions and antitumor activity characteristics in various in vitro and in vivo systems. The safety and tolerability profile of IMM40H were evaluated through single and repeated administration in cynomolgus monkeys. In vitro cell-based assays demonstrated that IMM40H had considerably stronger CD70-binding affinity than competitor anti-CD70 antibodies, including cusatuzumab, which enabled it to block the interaction of between CD70 and CD27 more effectively. IMM40H also exhibited potent Fc-dependent effector functions (ADCC/CDC/ADCP), and could make a strong immune attack on tumor cells and enhance therapeutic efficacy. Preclinical findings showed that IMM40H had potent antitumor activity in multiple myeloma U266B1 xenograft model, and could eradicate subcutaneously established tumors at a low dose of 0.3 mg/kg. IMM40H (0.3 mg/kg) showed therapeutic effects faster than cusatuzumab (1 mg/kg). A strong synergistic effect between IMM01 (SIRPα-Fc fusion protein) and IMM40H was recorded in Burkitt's lymphoma Raji and renal carcinoma cell A498 tumor models. In cynomolgus monkeys, the highest non-severely toxic dose (HNSTD) for repeat-dose toxicity was up to 30 mg/kg, while the maximum tolerated dose (MTD) for single-dose toxicity was up to 100 mg/kg, confirming that IMM40H had a good safety and tolerability profile. IMM40H is a high-affinity humanized IgG1 specifically targeting the CD70 monoclonal antibody with enhanced Fc-dependent activities. IMM40H has a dual mechanism of action: inducing cytotoxicity against CD70+ tumor cells via various effector functions (ADCC, ADCP and CDC) and obstructs the proliferation and activation of Tregs by inhibiting CD70/CD27 signaling.

Regulatory Mechanism of Mangiferin Combined with Bortezomib on Malignant Biological Behavior of Burkitt Lymphoma and Its Effect on Expression of CXC Chemokine Receptors

To analyze the effects of mangiferin combined with bortezomib on the proliferation, invasion, apoptosis and autophagy of human Burkitt lymphoma Raji cells, as well as the expression of CXC chemokine receptors (CXCRs) family, and explore the molecular mechanism between them to provide scientific basis for basic research and clinical work of Burkitt lymphoma. Raji cells were intervened with different concentrations of mangiferin and bortezomib alone or in combination, then cell proliferation was detected by CCK-8 assay, cell invasion ability was detected by Transwell chamber method, cell apoptosis was detected by Annexin V/PI double-staining flow cytometry, apoptosis, autophagy and Akt/mTOR pathway protein expression were detected by Western blot, and the expression changes of CXCR family was detected by real-time quantitative PCR (RT-qPCR). Different concentrations of mangiferin intervened Raji cells for different time could inhibit cell viability in a concentration- and time-dependent manner (r =-0.682, r =-0.836). When Raji cells were intervened by combination of mangiferin and bortezomib, compared with single drug group, the proliferation and invasion abilities were significantly decreased, while the apoptosis level was significantly increased (P <0.01). Mangiferin combined with bortezomib could significantly up-regulate the expression of pro-apoptotic protein Bax and down-regulate the expression of anti-apoptotic protein Bcl-2 after intervention in Raji cells. Caspase-3 was also hydrolyzed and activated, and then induced the apoptosis of Raji cells. Mangiferin combined with bortezomib could up-regulate the expression of LC3Ⅱ protein in Raji cells, and the ratio of LC3Ⅱ/LC3Ⅰ in cells was significantly up-regulated compared with single drug or control group (P <0.01). Mangiferin combined with bortezomib could significantly inhibit the phosphorylation levels of Akt and mTOR, inhibit the proliferation and invasion of Raji cells by inhibiting Akt/mTOR pathway, and induce cell autophagy and apoptosis. Mangiferin and bortezomib could down-regulate the expressions of CXCR4 and CXCR7 mRNA after single-agent intervention in Raji cells, and the down-regulations of CXCR4 and CXCR7 mRNA expression were more significant when the two drugs were combined (P <0.01). Mangiferin alone or combined with bortezomib had no significant effect on CXCR5 mRNA expression in Raji cells (P >0.05), while the combination of the two drugs could down-regulate the expression of CXCR3 (P <0.05). Mangiferin combined with bortezomib can synergistically inhibit the proliferation and invasion of Raji cells, and induce autophagy and apoptosis. The mechanism may be related to the inhibition of Akt/mTOR signaling pathway, down-regulation of anti-apoptotic protein Bcl-2 and up-regulation of pro-apoptotic protein Bax, and the inhibition of the expression of CXCR family.

Bioymifi suppresses Burkitt’s lymphoma cell line proliferation via cell cycle arrest and apoptosis

Purpose: To find a new treatment alternative for Burkitt’s lymphoma, one of the B cell-derived non-Hodgkin's lymphomas that has an extremely aggressive growth profile with a high mortality rate.&#x0D; Methods: Bioymifi (death receptor agonist) was administered at various doses to Human Burkitt's Lymphoma Raji cell lines, and cell proliferation was evaluated using XTT analysis. Apoptosis induction and cell cycle analysis were assessed using flow cytometry.&#x0D; Results: Bioymifi exhibited good cytotoxicity in Raji cell lines with IC50 value of 29.5 μM. Furthermore, results of apoptosis induction and cell cycle analysis indicated that Bioymifi had potent anti-proliferative activity through sub-S cell cycle arrest and stimulation of apoptosis (p &lt; 0.05).&#x0D; Conclusion: Bioymifi has anticancer potentials against Human Burkitt's Lymphoma Raji cell lines. Further pharmacological studies will be required to establish the mechanism of action.

Bryostatin Activates CAR T-Cell Antigen-Non-Specific Killing (CTAK), and CAR-T NK-Like Killing for Pre-B ALL, While Blocking Cytolysis of a Burkitt Lymphoma Cell Line.

The advent of CAR-T cell therapy has changed the face of clinical care for relapsed and refractory pre-B-acute lymphocytic leukemia (B-ALL) and lymphoma. Although curative responses are reported, long-term cures remain below 50%. Different CAR T-cell leukemia targets appear to have different mechanisms of CAR-T escape. For CD22, therapeutic evasion is linked to down-modulation of the number CD22 proteins expressed on the extracellular aspect of the leukemia cell plasma membrane. Recently, pharmacologic agents known to induce cellular differentiation or epigenetic modification of leukemia have been shown to impact CD22 and CD19 expression levels on B-ALL, and thereby increase sensitivity to CAR-T mediated cytolysis. We explored the impact of epigenetic modifiers and differentiation agents on leukemia cell lines of B cell origin, as well as normal B cells. We confirmed the activity of bryostatin to increase CD22 expression on model cell lines. However, bryostatin does not change CD22 levels on normal B cells. Furthermore, bryostatin inhibited CAR-T mediated cytolysis of the Raji Burkitt lymphoma cell line. Bryostatin increased the cytolysis by CD22 CAR-T for B-ALL cell lines by at least three mechanisms: 1) the previously reported increase in CD22 target cell numbers on the cell surface, 2) the induction of NK ligands, and 3) the induction of ligands that sensitize leukemia cells to activated T cell antigen-non-specific killing. The opposite effect was seen for Burkitt lymphoma, which arises from a more mature B cell lineage. These findings should caution investigators against a universal application of agents shown to increase killing of leukemia target cells by CAR-T in a specific disease class, and highlights that activation of non-CAR-mediated killing by activated T cells may play a significant role in the control of disease. We have termed the killing of leukemia targets, by a set of cell-surface receptors that does not overlap with NK-like killing “CTAK,” CAR-T Cell antigen-non-specific killing.

Construction and Characterization of 1F5 Chimeric Anti-CD20 Monoclonal Antibodies: an Efficient Splicing by Overlap Extension-polymerase Chain Reaction Technique

Despite the unparalleled success of anti-CD20-targeted immunotherapy, the currently available mAbs are not sufficiently efficacious in the treatment of lymphoma. 1F5 is one of a panel of anti-CD20 mAbs that was used in the B-cell lymphoma serotherapy. Despite the efficacy of murine 1F5 mAbs in lymphoma patients, the 1F5 chimeric antibodies with human effector functionality are yet to be approved and widely used in the treatment of lymphoma. In this study, the conversion of 1F5 mAb from mouse IgG2a to human-mouse chimeric IgG1 was achieved and the chimeric antibody was partially characterized. We constructed the 1F5 chimeric mouse-human anti-CD20 antibody genes using an efficient Splicing by overlap extension-polymerase chain reaction (SOE-PCR) technique and cloned the chimeric heavy and light genes in pBudCE4.1 mammalian expression vector, followed by purification of the expressed chimeric 1F5 mAbs using affinity chromatography. Our investigation also included the biological properties of purified chimeric antibodies. The generated 1F5 chimeric mAbs mediate complement-dependent cytotoxicity (CDC) and antibody-dependent cellular cytotoxicity (ADCC) against Raji and Daudi Burkitt's lymphoma cell lines, which were comparable with rituximab and exhibit superior reduction in cell viability in vitro, compared to rituximab. The current study indicated that the generated chimeric 1F5 mAbs has potential CDC and ADCC activity which was comparable with rituximab whereas it exhibits a superior reduction in cell viability, compared to rituximab. Our work contributes to future studies involving in vivo biological functions and the application of the 1F5 chimeric antibody.

Nicotinamide adenine dinucleotide phosphate oxidase inhibitor induces apoptosis on Epstein-Barr virus positive B lymphoma cells.

Over-expression of nicotinamide adenine dinucleotide phosphate oxidase (Nox) isoform enzymes was recently reported in various cancers including Burkitt’s lymphoma (BL). However, the functions of Nox isoform enzymes in BL remain poorly understood. In this study, Nox isoform expression and the effects of a Nox-specific inhibitor were evaluated in Epstein-Barr virus (EBV)-positive Raji BL cells in comparison with EBV-negative Ramos BL cells. To evaluate Nox enzyme expression in Raji and Ramos BL cells, polymerase chain reaction (PCR) and western blot analysis were performed. To verify the intracellular signaling mechanism of the Nox inhibitor-induced apoptosis of Raji cells, WST-1 assay, trypan blue exclusion method, flow cytometry, PCR, western blotting, and bromodeoxyuridine staining were conducted. Experiments using the pan-caspase inhibitor z-VAD, reactive oxygen species scavenger N-acetyl-L-cysteine (NAC), and Bim inhibitor 1 were performed. PCR and western blot results showed that Nox isoform enzymes were highly expressed in EBV-positive BL Raji cells compared with EBV-negative BL Ramos cells. The Nox2 inhibitor induced apoptosis of Raji cells in time- and dose-dependent manners. The Nox2 inhibitor also caused up-regulation of Bim and Noxa, down-regulation of Mcl-1, translocation of Bax, release of cytochrome c, and caspase cascade activation, resulting in apoptosis. Furthermore, z-VAD, NAC, and BI-1 effectively blocked the Nox2 inhibitor-induced apoptosis of Raji cells. Taken together, these results provide a novel insight into the mechanism of Nox inhibitor-induced apoptosis and evidence for Nox as a therapeutic target to treat EBV-positive malignancies.

A specific inhibitor of polo-like kinase 1, GSK461364A, suppresses proliferation of Raji Burkitt's lymphoma cells through mediating cell cycle arrest, DNA damage, and apoptosis

Polo-like kinase 1 (PLK1) is a prominent mediatory player during the cell cycle, mitosis, and cytokinesis in eukaryotic cells. Besides its physiological roles, PLK1 expression is upregulated in a wide range of human malignant tumors and its overexpression worsens prognosis, therefore, specific inhibition of PLK1 in tumor cells is a fascinating approach for the development of novel chemotherapeutics. The present study elucidated the potential cytotoxic effects of a PLK1 inhibitor, GSK461364A, in five cancer cell lines including Raji, K562, PC3, MCF-7, MDA-MB-231, along with noncancerous L929 cells by XTT assay. The cells were treated for 24 h with GSK461364A at different concentrations ranged between 0.5 and 40 μM and significant cytotoxicity was observed in all treated groups with the IC50 values between 2.36 and 4.08 μM. GSK461364A was also found to be safer with lower cytotoxicity against L929 cells and the IC50 value was found to be greater than 40 μM. Raji cells were identified as the most sensitive cell line against GSK461364A with the lowest IC50 values, hence it was selected for further studies to evaluate the underlying mechanism of cytotoxic activity. The treatment of Raji cells with GSK461364A caused a cell cycle arrest at the G2/M phase, also altered TOS, which is an indicator of oxidative stress, and DNA damage response, significantly. The Annexin V binding assay revealed that GSK461364A treatment significantly increased in the percentage of early and late apoptotic cells. Fluorescence imaging also showed that GSK461364A treatment significantly induced apoptosis of Raji cells. The apoptotic effect of the compound has also been confirmed by increased expressions of Bax and cleaved caspase 3 and along with the decreased expression of BCL-2. The results demonstrated that GSK461364A induced anticancer effects which was mainly promoted by cell cycle arrest, oxidative stress, DNA damage, and finally apoptosis in Burkitt's lymphoma cells. Taken together, the present results emphasized that GSK461364A could be a useful therapeutic agent in patients with Burkitt's lymphoma. However, further studies are required to consolidate the anticancer activity of this promising compound.

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.

Catfish egg lectin affects influx and efflux rates of sunitinib in human cervical carcinoma HeLa cells.

New treatment protocols are aiming to reduce the dose of the multitargeted tyrosine kinase inhibitor sunitinib, as sunitinib elicits many adverse effects depending on its dosage. Silurus asotus egg lectin (SAL) has been reported to enhance the incorporation of propidium iodide as well as doxorubicin into Burkitt's lymphoma Raji cells through binding to globotriaosylceramide (Gb3) on the cell surface. The objective of this study was to examine whether SAL enhances the cytotoxic effect of sunitinib in Gb3-expressing HeLa cells. Although the treatment with SAL delayed the cell growth and enhanced the propidium iodide uptake, cell death accompanied by membrane collapse was not observed. The viability of sunitinib-treated HeLa cells was significantly reduced when the treatment occurred in combination with SAL compared to their separate usage. Sunitinib uptake significantly increased for 30min in SAL-treated cells, and this increment was almost completely abolished by the addition of L-rhamnose, a hapten sugar of SAL, but not by D-glucose. After removal of SU from the medium, the intracellular sunitinib level in SAL-treated cells was higher than in untreated cells for 24h, which was not observed in Gb3-deficient HeLa cells. Furthermore, we observed that SAL promoted the formation of lysosome-like structures, which are LAMP1 positive but not acidic in HeLa cells, which can trap sunitinib. Interestingly, SAL-induced vacuolation in HeLa cells was not observed in another Gb3 positive Raji cells. Our findings suggest that SAL/Gb3 interaction promoted sunitinib uptake and suppressed sunitinib excretion and that sunitinib efficiently exerted cytotoxicity against HeLa cells.

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.

Functional Characterization of Gamma Delta T Cells and Allogeneic Activated NK Cells As Effector Cells for Tafasitamab (MOR208)

Introduction Tafasitamab (MOR208) is an Fc-enhanced, humanized, monoclonal antibody that binds to the human B cell surface antigen CD19. CD19 is broadly and homogeneously expressed across different B cell malignancies, including diffuse large B cell lymphoma (DLBCL), and amplifies B cell receptor signaling and induces tumor cell proliferation. Tafasitamab is currently in clinical development in patients with relapsed or refractory DLBCL in combination with the immunomodulatory drug lenalidomide (L-MIND study) and the chemotherapeutic agent bendamustine (B-MIND study). The modes of action of tafasitamab comprise antibody-dependent cellular cytotoxicity (ADCC), antibody-dependent cellular phagocytosis (ADCP) and direct cytotoxicity (apoptosis). Tafasitamab carries two amino acid exchanges in the Fc region to increase its affinity to Fcγ receptors, including FcγRIIIa. FcγRIIIa plays a key role in mediating ADCC and is expressed on the surface of natural killer (NK) cells, as well as the majority of γδ T cells. MG4101 (a novel therapeutic agent consisting of cryopreserved, ex vivo-expanded, highly activated NK cells) has demonstrated potent anticancer activity in preclinical in vitro and in vivo studies. Currently, MG4101 is in clinical development in patients with malignant lymphoma and advanced solid tumors. Here, we have characterized two FcγRIIIa receptor-expressing cell types, γδ T cells and NK cells (MG4101), as effector cells for tafasitamab in vitro and explored the concept of supplementing MG4101 during tafasitamab therapy using disseminated in vivo models of non-Hodgkin's lymphoma. Methods γδ T cells (CD3+/γδ T cell receptor+) were derived from different donors by stimulation of peripheral blood mononuclear cells with zoledronate/IL-2 for 9-10 days. These were applied as effector cells in in vitro ADCC assays with tafasitamab in Mino and Jeko-1 mantle cell lymphoma (MCL) cell lines, as well as primary patient-derived chronic lymphocytic lymphoma (CLL) and MCL cells. Further, effector cell activity of MG4101 was assessed using tafasitamab-mediated ADCC assays in Raji and Ramos Burkitt's lymphoma cells. The concept of combining tafasitamab with allogeneic effector cell therapy in vivo was studied in two therapeutic survival models of disseminated lymphoma in SCID mice. In the Raji model, tafasitamab (0.05 µg/mouse) was given on Day 1 after intravenous (IV) tumor inoculation, while MG4101 (2x107 cells/mouse) was given on Days 1, 3, 6, 8 and 10. In the Ramos model, tafasitamab (10 mg/kg) and MG4101 (2x107 cells/mouse) were applied twice weekly for 3 weeks starting on Days 3 and 4, respectively, after IV tumor inoculation. Results Tafasitamab in combination with γδ T cells showed distinctly increased ADCC in Mino and Jeko-1 target cells in vitro, compared with a negative control IgG1 antibody. ADCC assays with patient-derived CLL and MCL target cells confirmed tafasitamab-mediated cytotoxic activity and demonstrated a clear enhancement in activity compared with the non-Fc-enhanced version of tafasitamab that was unable to induce substantial cytotoxicity. In vitro ADCC assays with tafasitamab and MG4101 on Raji and Ramos cell lines confirmed potent effector cell activity of the ex vivo-expanded, cryopreserved, allogeneic NK cells. In the disseminated Raji survival model, combination therapy with a single low dose of tafasitamab (0.05 µg) and MG4101 resulted in a distinct increase in survival of the mice with an increased life span (ILS) of 100% compared with monotherapy (ILS of 57% for tafasitamab and 50% for MG4101). Of note, the combination demonstrated a substantial and more than additive enhancement in survival in the more therapeutic Ramos survival model (Figure 1). Combination therapy with tafasitamab (10 mg/kg) and MG4101 NK cells resulted in superior antitumor activity (ILS of 103%) compared with either tafasitamab monotherapy (ILS of 49%) or MG4101 alone (ILS of 25%). Conclusions FcγRIIIa-expressing immune cell types, including NK cells and γδ T cells, are potent effector cells for tafasitamab-mediated ADCC. Combination therapy with tafasitamab and allogeneic MG4101 NK cells in vivo demonstrated a more than additive survival benefit compared with tafasitamab or MG4101 monotherapy in a disseminated therapeutic lymphoma model. Combination of tafasitamab supplemented with immune effector cells could represent a promising new approach for lymphoma therapy. Disclosures Her: GC LabCell: Employment, Patents &amp; Royalties. Cho:GC LabCell: Employment, Patents &amp; Royalties. Hwang:GC LabCell: Employment, Equity Ownership, Patents &amp; Royalties. Boxhammer:MorphoSys AG: Employment, Patents &amp; Royalties. Steidl:MorphoSys AG: Employment. Patra:MorphoSys AG: Employment. Schanzer:MorphoSys AG: Employment. Endell:MorphoSys AG: Employment, Patents &amp; Royalties.

Knockdown of long non-coding RNA PVT1 inhibits the proliferation of Raji cells through cell cycle regulation.

Long non-coding RNA plasmacytoma variant translocation 1 (PVT1) has been reported to be associated with oncogenesis. However, the functional role of PVT1 in Burkitt lymphoma has not yet been addressed. The purpose of the present study was to investigate the effect of PVT1 knockdown by small interfering RNA (siRNA) on the proliferation of Burkitt lymphoma Raji cells and to explore its possible mechanism of action. An effective siRNA targeting PVT1 was screened and the corresponding short hairpin RNA (shRNA) was reconstructed into a lentiviral vector. Cell proliferation and cell cycle distribution were assessed by Cell Counting kit-8 assay and flow cytometry, respectively. Protein expression levels of c-Myc, cyclin-dependent kinase inhibitor1A (CDKN1A, P21) and cyclin E1 (CCNE1) were detected by western blotting. A polymerase chain reaction (PCR) array was used to analyse the expression of genes associated with the cell cycle. PVT1 knockdown markedly suppressed proliferation, and induced cell cycle arrest at the G0/G1 phase in Raji cells. Protein expression levels of c-Myc and CCNE1 were reduced, whereas P21 protein expression was markedly increased following downregulation of PVT1 in Raji cells. The cell cycle PCR array revealed that 54 genes were upregulated and 26 genes were downregulated in Raji cells following PVT1 knockdown. Reverse transcription-quantitative PCR demonstrated that cyclin G2 (CCNG2), CDKN1A, Retinoblastoma-like 2 (RBL2, p130), HUS1 checkpoint homolog, cyclin dependent kinase inhibitor 3 (CDKN3) and cyclin dependent kinase inhibitor 1B (CDKN1B) expression were upregulated, whereas the expression levels of CCNE1, cyclin D1 (CCND1) and cell division cycle 20 (CDC20) were downregulated in Raji cells with PVT1 knockdown. In conclusion, PVT1 knockdown may inhibit the proliferation of Raji cells by arresting cells in G0/G1 phase. Furthermore, inhibition of cell proliferation may be associated with a reduction inc-Myc expression and alterations in the expression levels of cell cycle-associated genes.

Effects of enhancer of Zeste homolog 2 inhibitor GSK126 on the cell proliferation and apoptosis of acute leukemia and lymphoma cells in vitro

Objective To investigate the effects of a novel enhancer of Zeste homolog 2 (EZH2) inhibitor GSK126 in vitro on the cell proliferation and apoptosis in acute leukemia and lymphoma cells. Methods CCK-8 assay was used to measure the effects of GSK126 with different concentrations and time on the cell proliferation in human T-cell acute lymphoblastic leukemia (T-ALL) CEM cell line, acute monocytic leukemia U937 cell line and Burkitt lymphoma Raji cell line. Annexin V/PI double staining method was used to determine the effects of GSK126 on the cell apoptosis. The effect of GSK126 on the mRNA levels of EZH2, bcl-2 and bcl-xL were measured by using quantitative polymerase chain reaction (qPCR). Results After the function for 24 h, 48 h and 72 h, compared to the negative control group (0 μmol/L), 5, 10, 15, 20, 25 μmol/L GSK126 treatment showed a significant dose-and time-dependent cell proliferation suppression in CEM cells; 5, 10, 15, 20 μmol/L GSK126 treatment showed a significant dose-and time-dependent cell proliferation suppression in U937 cells; 5, 10, 15, 20, 25, 30 μmol/L GSK126 treatment showed a significant time-dependent suppression in Raji cells (all P < 0.05). The 48 h 50% inhibitory concentration (IC50) of GSK126 on CEM, U937, Raji cells was (13.46±0.83), (11.65±1.02), (15.00±0.19) μmol/L, respectively. GSK126 treatment with the doses of 8, 12 and 16 μmol/L promoted the apoptosis of CEM and U937 cells compared with the negative control group (0 μmol/L), and there were statistical differences in the apoptosis rate (F = 167.995, P < 0.01; F = 158.400, P < 0.01). In Raji cells, only 16 μmol/L GSK126 treatment had a higher cell apoptosis rate compared with the control group (t = 47.998, P < 0.05). Furthermore, 8, 12 and 16 μmol/L GSK126 treatment suppressed the expressions of EZH2 mRNA in CEM, U937 and Raji cells compared with the control group (F = 82.035, P < 0.01; F = 252.712, P < 0.01; F = 690.536, P < 0.01), and the expressions of bcl-2 and bcl-xL mRNA (bcl-2: F = 1 900.525, P < 0.01; F = 431.324, P < 0.01; F = 216.184, P < 0.01; bcl-xL: F = 256.751, P < 0.01; F = 147.019, P < 0.01; F = 209.325, P < 0.01). Conclusion EZH2 plays an important role in the occurrence and development of acute leukemia and lymphoma. GSK126 as a high selective inhibitor of EZH2 might be a potential new drug in treatment of hematological malignancies. Key words: Hematologic neoplasms; Cell proliferation; Apoptosis; Enhancer of Zeste homolog 2; GSK126

Anti-human SIRPα antibody is a new tool for cancer immunotherapy.

Interaction of signal regulatory protein α (SIRPα) expressed on the surface of macrophages with its ligand CD47 expressed on target cells negatively regulates phagocytosis of the latter cells by the former. We recently showed that blocking Abs to mouse SIRPα enhanced both the Ab‐dependent cellular phagocytosis (ADCP) activity of mouse macrophages for Burkitt's lymphoma Raji cells opsonized with an Ab to CD20 (rituximab) in vitro as well as the inhibitory effect of rituximab on the growth of tumors formed by Raji cells in nonobese diabetic (NOD)/SCID mice. However, the effects of blocking Abs to human SIRPα in preclinical cancer models have remained unclear given that such Abs have failed to interact with endogenous SIRPα expressed on macrophages of immunodeficient mice. With the use of Rag2−/−γc −/− mice harboring a transgene for human SIRPα under the control of human regulatory elements (hSIRPα‐DKO mice), we here show that a blocking Ab to human SIRPα significantly enhanced the ADCP activity of macrophages derived from these mice for human cancer cells. The anti‐human SIRPα Ab also markedly enhanced the inhibitory effect of rituximab on the growth of tumors formed by Raji cells in hSIRPα‐DKO mice. Our results thus suggest that the combination of Abs to human SIRPα with therapeutic Abs specific for tumor antigens warrants further investigation for potential application to cancer immunotherapy. In addition, humanized mice, such as hSIRPα‐DKO mice, should prove useful for validation of the antitumor effects of checkpoint inhibitors before testing in clinical trials.

Targeted downregulation of MYC through G‐quadruplex stabilization: a novel therapeutic approach for breast cancer and lymphoma

MYC, a basic helix‐loop‐helix/leucine zipper transcription factor, is an enigmatic protein with over 30,000 potential binding sites in the human genome, 10–15% of which are generally bound at any one time. It was one of the first proto‐oncogenes to be described and is ultimately disregulated in most tumor types and stages, including estrogen receptor (ER)‐positive breast cancer and non‐Hodgkin's lymphoma. Decreased MYC expression has been shown to significantly impact tumor viability while offering a notable therapeutic window and overall safe profile. As we and other groups have shown, stabilization of the higher order genomic structure – the G‐quadruplex (G4) – in the MYC promoter is an established approach to decreasing transcription and facilitating tumor lysis. We have pioneered a DNA interference (DNAi) approach to take advantage of the “druggability” of the DNA structure, and have previously demonstrated the specificity and efficacy of oligonucleotide (ODN)‐mediated stabilization of the predominant G4 structure within the MYC promoter. In the current report, we further optimize the DNAi, including the evaluation of different ODN lengths, for specifically stabilizing the MYC promoter G4 (as demonstrated by EMSA and chemical footprinting) and modulating promoter activity. The lead DNAi (A‐18) demonstrates anti‐cancer activity in RAJI and CA46 Burkitt's lymphoma cells, and in ER+ MCF‐7 breast cancer cells. Moreover, A‐18 enters the nuclei of cancer cells, where it binds the G4‐forming region of the MYC promoter, in an inducible manner. This clamp has potential as a novel DNAi therapeutic. It is being further explored for its ability to sensitize cancer cells to chemotherapeutic agents, and its formulation for delivery in nanotherapeutic vehicles.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

FLAG-tagged CD19-specific CAR-T cells eliminate CD19-bearing solid tumor cells in vitro and in vivo.

Autologous T cells expressing chimeric antigen receptors (CARs) specific for CD19 have demonstrated remarkable efficacy as therapeutics for B cell malignancies. In the present study, we generated FLAG-tagged CD19-specific CAR-T cells (CD19-FLAG) and compared them to their non-tagged counterparts for their effects on solid and hematological cancer cells in vitro and in vivo. For solid tumors, we used HeLa cervical carcinoma cells engineered to overexpress CD19 (HeLa-CD19), and for hematological cancer we used Raji Burkitt's lymphoma cells, which endogenously express CD19. Like non-tagged CD19 CAR-T cells, CD19-FLAG CAR-T cells expanded in culture >100-fold and exhibited potent cytolytic activity against both HeLa-CD19 and Raji cells in vitro. CD19-FLAG CAR-T cells also secreted significantly more IFN-gamma and IL-2 than the control T cells. In vivo, CD19-FLAG CAR-T cells significantly blocked the growth of HeLa-CD19 solid tumors, increased tumor cleaved caspase-3 levels, and expanded systemically. CD19-FLAG CAR-T cells also significantly reduced Raji tumor burden and extended mouse survival. These results demonstrate the strong efficacy of FLAG-tagged CD19 CAR-T cells in solid and hematological cancer models.

Drug-free macromolecular therapeutics: Impact of structure on induction of apoptosis in Raji B cells

Recently, we developed a new paradigm in macromolecular therapeutics that avoids the use of low molecular weight drugs. The activity of the “drug-free macromolecular therapeutics” is based on the biorecognition of complementary motifs at cell surface resulting in receptor crosslinking and apoptosis induction. The system is composed of two nanoconjugates: (1) a single-stranded morpholino oligonucleotide (MORF1) attached to an anti-CD20 Fab′ fragment (Fab′-MORF1); (2) multiple copies of complementary oligonucleotide MORF2 grafted to a linear polymer of N-(2-hydroxypropyl)methacrylamide (HPMA) – P-(MORF2)x. The two conjugates crosslink CD20 antigens via MORF1-MORF2 hybridization at the surface of CD20+ malignant B-cells and induce apoptosis. Preclinical studies in a murine model of human non-Hodgkin's lymphoma showed cancer cells eradication and long-term survivors. The aim of this study was to determine the relationship between the detailed structure of the nanoconjugates and apoptosis induction in Raji cells to allow system optimization. The factors studied include the length of the MORF sequence, the valence of P-(MORF2)x (varying x), molecular weight of P-(MORF2)x, incorporation of a miniPEG spacer between Fab′ and MORF1 and between polymer backbone and pendant MORF2, and comparison of two Fab′ fragments, one from 1F5 antibody (Fab′1F5), the other from Rituximab (Fab′RTX). The results of apoptosis induction in human Burkitt's B-cell non-Hodgkin's lymphoma (NHL) Raji cells as determined using three apoptotic assays (Annexin V, Caspase 3, and TUNEL) indicated that: a) An improvement of apoptotic activity was observed for a 28 base pair MORF sequence when compared to MORFs composed of 20 and 25 base pairs. The differences depended on type of assay, concentration and exposure schedule (consecutive vs. premixed). b) The higher the valence of P-(MORF2)x the higher the levels of apoptosis. c) Higher molecular weight of P-(MORF2)x induced higher levels of apoptosis. d) A miniPEG8 spacer was effective in enhancing apoptotic levels in contrast to a miniPEG2 spacer. e) There was not a statistically significant difference when comparing Fab′1F5-MORF1 with Fab′RTX-MORF1.

Lysosomal Disruption Augments Obinutuzumab-Induced Direct Cell Death

Anti-CD20 mononclonal antibodies (mAbs) have a well-established role in the treatment of B-cell lymphoid malignancies. In addition to classic Fc-dependent mechanisms, including antibody-dependent and complement-mediated cytotoxicity, the so-called type II mAbs induce direct cell death. It has been shown that obinutuzumab, without Fc-crosslinking agents or effector cells, triggers non-apoptotic, lysosomal-dependent programmed cell death (PCD). The mechanism of PCD is characterized by actin reorganization, followed by permeabilization of the lysosomal membrane and subsequent generation of reactive oxygen species (ROS) through NADPH oxidase. Although, mechanisms of PCD are well-described, little is known about factors influencing sensitivity of malignant B-cells to obinutuzumab-mediated direct cell killing. Strategies to improve PCD could be potentially exploited to eliminate malignant cells, which are refractory to conventional immunotherapy. In this study, we aimed to investigate the influence of lysosomotropic agent, chloroquine, on the efficacy of obinutuzumab-mediated cytotoxicity. As PCD is dependent on lysosomal destabilization, we hypothesized that combination of obinutuzumab with lysosome-destabilizing agent would result in increased cell death.In our study, we used a Burkitt lymphoma Raji cell line that is widely employed as a model to assess the efficacy of obinutuzumab. Raji cells were incubated with obinutuzumab, alone or in combination with increasing concentrations of chloroquine, followed by annexin V/PI staining. Chloroquine, significantly increased direct cell death induced by obinutuzumab, without being toxic alone. Those observations were further corroborated by cell staining with other viability dies - TO-PRO-3 iodide and 7-AAD.The efficacy of the tested combination was completely abrogated by cytochalasin D - an inhibitor of actin polymerization and concanamycin A - inhibitor of vacuolar ATPases that activate acidic vacuoles including lysosomes, suggesting that chloroquine and obinutuzumab share a common mechanism of action. Since chloroquine has been reported to promote ROS generation, we analyzed the production of ROS with DCFDA staining. We observed that chloroquine potentiated the oxidative effect of obinutuzumab. Consistently, the effect of the combination was completely abrogated by a cell-permeable ROS scavenger - Tiron. As obinutuzumab has been shown to induce ROS production via activation of NADPH oxidase, we investigated the influence of NADPH oxidase inhibitor - diphenylene iodonium (DPI) on the combination's efficacy. DPI only partially reversed the effect of obinutuzumab, while strongly decreasing the effect of the combination.Altogether, we show for the first time that chloroquine sensitizes cell to lysosomal cell death induced by obinutuzumab. The results of our study provide a strong rationale for combining obinutuzumab with lysosomotropic agents. These findings may have particular importance given the fact that another lysomotropic agent - siramesine has recently been shown to induce selective cytotoxicity in chronic lymphocytic leukemia (CLL). DisclosuresNo relevant conflicts of interest to declare.

Ouabain induces apoptosis and autophagy in Burkitt's lymphoma Raji cells.

The steroid Na+/K+-ATPase blocker ouabain has been shown to exhibit cytotoxic effects in various tumor cell systems. This study aimed to determine the effects of ouabain on Burkitt's lymphoma Raji cells. Ouabain treatment of Raji cells significantly inhibited cell proliferation in a dose-dependent manner and increased the morphological changes associated with apoptosis. Additionally, increased numbers of both early and late apoptotic cells were observed by annexin V-FITC/PI flow cytometry assay. Increased levels of caspase-3 and cleaved-caspase-3, higher Bax activity and decreased expression of the anti-apoptotic protein Bcl-2 were detected in ouabain-treated Raji cells. Vacuole accumulation was also observed in transmission electron microscope (TEM) images of ouabain-treated Raji cells, indicating that these cells were undergoing autophagy. Expression of the autophagy-related proteins LC3-II and Beclin-1 was upregulated in ouabain-treated Raji cells. These results suggest that ouabain may promote cell death in Raji cells by inducing pathways associated with apoptosis and autophagy. Our study also provides novel evidence that ouabain may be an effective agent for treating Burkitt's lymphoma.