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Burkitt's lymphoma (BL) has a particularly extremely poor prognosis and the fastest growth rate among human tumors, and the development of new drugs for the treatment of BL is urgently needed. In this study, the cytotoxic properties of 3,7-bis(3,5-dimethylphenyl)-aaptamine (AP-51), a new semisynthetic alkaloid derived from the marine natural product aapatamine, were investigated using BL cell lines. Our results showed that AP-51 inhibited the proliferation of Daudi and Raji cells with IC 50 values of 3.48 and 2.07 μM, respectively. Flow cytometry and Western blot analyses showed that AP-51 initiated G0/G1 phase arrest by modulating the expression of cyclin-dependent kinases (CDKs). AP-51 also induced apoptosis, as demonstrated by nuclear fragmentation, downregulation of BCL-XL and Mcl-1, and upregulation of cleaved caspase-9, cleaved caspase-3, cleaved-PARP, and cytochrome c , the markers of apoptosis regulated via the mitochondrial pathway. When it comes to mitochondria, AP-51 treatment also significantly increased the levels of intracellular mitochondrial superoxide, decreased ATP content, and reduced the expression of ATP synthase, as well as the expression of the mitochondrial respiratory chain complexes. Finally, AP-51 treatment significantly inhibited the PI3K/AKT/mTOR signaling pathway, which was shown to be associated with the induction of apoptosis. Collectively, these findings indicated that AP-51 initiated cell cycle arrest, induced apoptosis, caused mitochondrial dysfunction, and decreased the phosphorylation of PI3K/AKT/mTOR signaling pathway-related proteins and the protein levels of C-MYC, suggesting that AP-51 has therapeutic potential as a possible treatment for Burkitt's lymphoma.

Increased expression levels of the Oct-1 transcription factor is considered to be one of the key markers of poor cancer prognosis. In addition to the ubiquitous Oct-1A isoform, which is found in all cells, there also exists a tissue-specific Oct-1L isoform, which is expressed in hematopoietic cells. Oct-1L increases cell resistance to different stresses and also regulates the expression of genes controlling differentiation of hematopoietic and immune system cells. The tissue-specific Oct-1L isoform levels are significantly increased in the B-cell lymphoblastoma Namalwa and Raji lines and the T-cell lymphoblastoma Jurkat line compared to normal B and T cells. Apparently, aberrant Oct-1L overexpression not only enhances stress resistance but also leads to the disruption of developmental pathways in the cells promoting their malignant transformation. We report here that targeted suppression of the tissue-specific Oct-1L isoform expression reduces the proliferation rate of Namalwa B-lymphoblastic Burkitt's lymphoma cells, significantly increases cell death rate under hypoxic conditions, and makes cells more sensitive to chemotherapeutic agents such as docetaxel and doxorubicin. These results indicate that targeted therapy aimed at the suppression of the Oct-1 isoforms with increased expression levels in tumor cells rather than the total Oct-1, thus avoiding the traumatic effects of total Oct-1 knockdown, may be promising. Selective suppression of Oct-1 isoforms is a promising strategy in the treatment of lymphoid tumors and may contribute to mitigating the disease course and increasing survival rates in cancer patients.

Burkitt's lymphoma (BL) is the fastest growing human tumor. Current treatment consists of a multiagent regimen of cytotoxic drugs with serious side effjects including tumor lysis, cardiotoxicity, hepatic impairment, neuropathy, myelosuppression, increased susceptibility to malignancy, and death. Furthermore, therapeutic interventions in areas of BL prevalence are not as feasible as in high-income countries. Therefore, there exists an urgent need to identify new therapies with a safer profile and improved accessibility. Triclosan (TCS), an antimicrobial used in personal care products and surgical scrubs, has gained considerable interest as an antitumor agent due to its interference with fatty acid synthesis. Here, we investigate the antitumor properties and associated molecular mechanisms of TCS in BL-derived BJAB cells. Dose-dependent cell death was observed following treatment with 10-100 µM TCS for 24 h, which was associated with membrane phospholipid scrambling, compromised permeability, and cell shrinkage. TCS-induced cell death was accompanied by elevated intracellular calcium, perturbed redox balance, chromatin condensation, and DNA fragmentation. TCS upregulated Bad expression and downregulated that of Bcl2. Moreover, caspase and JNK MAPK signaling were required for the full apoptotic activity of TCS. In conclusion, this report identifies TCS as an antitumor agent and provides new insights into the molecular mechanisms governing TCS-induced apoptosis in BL cells.

Burkitt lymphoma (BL) is a highly aggressive form of non-Hodgkin's B-cell lymphoma. Currently, multi-agent chemotherapy regimens are being used to significantly improve cure rates and achieve complete remissions in BL patients. However, drug resistance can often occur within 6 months in BL patients, contributing to poor prognosis. Mounting evidence suggests that cell adhesion-mediated drug resistance (CAM-DR), caused by the interaction between the bone marrow microenvironment and tumour cells may play an important role in drug resistance to chemotherapy. However, the molecular mechanism underlying CAM-DR in BL has not been identified yet. In this study, we investigated the molecular mechanism responsible for CAM-DR in BL cells. We also examined the therapeutic targets of CAM-DR in BL cells and found CD49d and CD49e to be the important adhesion molecules involved. However, CD49a, CD49b, CD11a, CD29, CD18, and CD61 were not found to be associated with CAM-DR in BL cells. Furthermore, we clarified that CD49d- and CD49e-mediated CAM-DR could be attributed to an increase in the expression of B cell leukemia-xL (Bcl-xL) and survivin proteins, and a decrease in the expression of Bcl-2 associated X (Bax), Bcl-2 interacting mediator (Bim) and p53 upregulated modulator of apoptosis (PUMA) proteins via nuclear factor kappaB (NF-κB) activation. In addition, bortezomib was found to overcome CAM-DR in BL cells by inhibiting NF-κB. Thus, bortezomib may have potential clinical applications in the treatment of CD49d- and CD49e-mediated CAM-DR in BL patients.

Shiga toxins (Stxs) expressed by the enterohaemorrhagic Escherichia coli and enteric Shigella dysenteriae 1 pathogens are protein synthesis inhibitors. Stxs have been shown to induce apoptosis via the activation of extrinsic and intrinsic pathways in many cell types (epithelial, endothelial, and B cells) but the link between the protein synthesis inhibition and caspase activation is still unclear. Endoplasmic reticulum (ER) stress induced by the inhibition of protein synthesis may be this missing link. Here, we show that the treatment of Burkitt lymphoma (BL) cells with verotoxin-1 (VT-1 or Stx1) consistently induced the ER stress response by activation of IRE1 and ATF6-two ER stress sensors-followed by increased expression of the transcription factor C/REB homologous protein (CHOP). However, our data suggest that, although ER stress is systematically induced by VT-1 in BL cells, its role in cell death appears to be cell specific and can be the opposite: ER stress may enhance VT-1-induced apoptosis through CHOP or play a protective role through ER-phagy, depending on the cell line. Several engineered Stxs are currently under investigation as potential anti-cancer agents. Our results suggest that a better understanding of the signaling pathways induced by Stxs is needed before using them in the clinic.

Background: Current chemotherapies for Burkitt lymphoma (BL) have dramatically improved its clinical outcome. However, chemoresistance can lead to chemotherapy failure and very poor prognosis; thus, novel strategies are urgently required for patients with drug-resistant BL. To investigate the mechanisms underlying drug resistance in BL, we established drug-resistant BL cell lines: HS-Sultan/ADM (adriamycin-resistant), HS-Sultan/VCR (vincristine-resistant), HS-Sultan/DEX (dexamethasone-resistant), and HS-Sultan/L-PAM (melphalan-resistant)., Methods: Drug transporter and survival factor expression were investigated the using western blotting and real time polymerase chain reaction. Cell survival was analyzed by trypan blue dye exclusion method., Results: The established cell lines acquired cross-resistance to adriamycin, vincristine, dexamethasone, and melphalan and exhibited 50% inhibitory concentration values 106-, 40-, 81-, and 45-fold higher than the parental cell lines, respectively. We found that protein and mRNA expression of MDR1 and Survivin were higher in drug-resistant BL cells than in the parent cells. Treatment with verapamil, an MDR1 inhibitor, or Survivin siRNA alongside each anti-cancer drug suppressed the proliferation of all drug-resistant BL cells. Src kinase activity was higher in all resistant cell lines than the parental cells; suppressing Src with dasatinib restored drug sensitivity by reducing MDR1 and Survivin expression., Conclusions: MDR1 and Survivin upregulation are responsible for resistance to conventional drugs and dasatinib can restore drug sensitivity by reducing MDR1 and Survivin expression in drug-resistant BL cells. Src inhibitors could therefore be a novel treatment strategy for patients with drug resistant BL.

Currently, there is no effective treatment for Burkitt's lymphoma in patients aged above 60 years, and thus research on effective treatment options for Burkitt's lymphoma has been gaining increasing attention. Artesunate has been identified as a novel effective growth suppressor in Burkitt's lymphoma. Here, we utilized molecular biology, transcriptome analysis, and other techniques to study artesunate-induced death of the Burkitt's lymphoma cells DAUDI and CA-46, the effect of artesunate on gene expression in DAUDI and CA-46 cells, and the effect of artesunate-induced ATF4-CHOP-CHAC1 pathway on ferroptosis. We also studied the inhibitory effects and ferroptosis induction of artesunate on CA-46 cells in mouse xenografts. Results showed that artesunate induced ferroptosis in DAUDI and CA-46 cells, as evidenced by the protective effect of liproxstatin-1, ferrostatin-1, and desferoxamine, resulting in an endoplasmic reticulum stress response, activation of the ATF4-CHOP-CHAC1 pathway enhanced ferroptosis in DAUDI and CA-46 cells. A mouse-transplanted tumor model showed that artesunate can inhibit the proliferation and induce ferroptosis of CA-46 cells in vivo. This study provides a novel perspective for the development of drugs against different types of Burkitt's lymphomas., (Copyright © 2019 Elsevier Inc. All rights reserved.)

Therapeutic targeting of initiating oncogenes is the mainstay of precision medicine. Considerable efforts have been expended toward silencing MYC, which drives many human cancers including Burkitt lymphomas (BL). Yet, the effects of MYC silencing on standard-of-care therapies are poorly understood. Here we found that inhibition of MYC transcription renders B-lymphoblastoid cells refractory to chemotherapeutic agents. This suggested that in the context of chemotherapy, stabilization of Myc protein could be more beneficial than its inactivation. We tested this hypothesis by pharmacologically inhibiting glycogen synthase kinase 3β (GSK-3β), which normally targets Myc for proteasomal degradation. We discovered that chemorefractory BL cell lines responded better to doxorubicin and other anti-cancer drugs when Myc was transiently stabilized. In vivo, GSK3 inhibitors (GSK3i) enhanced doxorubicin-induced apoptosis in BL patient-derived xenografts (BL-PDX), as well as in murine MYC-driven lymphoma allografts. This enhancement was accompanied by and required deregulation of several key genes acting in the extrinsic, death-receptor-mediated apoptotic pathway. Consistent with this mechanism of action, GSK3i also facilitated lymphoma cell killing by a death ligand TRAIL and by a death receptor agonist mapatumumab. Thus, GSK3i synergizes with both standard chemotherapeutics and direct engagers of death receptors and could improve outcomes in patients with refractory lymphomas.

Aim: Curcumin, a bioactive compound from turmeric, eliminates cancer stem cells (CSCs) in some cancers. This study evaluates the effects of curcumin on CSC markers in Burkitt lymphoma and acute myeloid leukemia cells., Methods: Cells were treated with increasing concentrations of curcumin, followed by an ALDEFLUOR assay, colony formation assay and western blot analysis for the CSC-associated proteins, Gli-1, Notch-1 and Cyclin D1., Results: Markers associated with CSCs were decreased in cells treated with curcumin. This included a decrease in the percentage of ALDH-positive cells, a decrease in colony formation and the downregulation of Gli-1, Notch-1 and Cyclin D1., Conclusion: These results indicate that curcumin decreased CSC markers in lymphoma/leukemia cells, potentially through inhibiting self-renewal.

Burkitt's lymphoma is a type of highly aggressive Non-Hodgkin's lymphoma. Although advanced Burkitt's lymphoma is responsive to high-intensity chemotherapy regimens, increasing systemic toxicity, tumor recurrence and metastasis significantly reduce the patient survival. Thus, it is important to investigate novel antitumor agents with safety and effectiveness. β-elemene shows anti-proliferative effect on cancer cells by triggering apoptosis through regulating several molecular signaling pathways. However, its role in the suppression of Burkitt's lymphoma has not yet been fully elucidated. The inhibitory effect of β-elemene in Burkitt's lymphoma was studied in vitro and in vivo, as well as the involved molecular mechanism. The results demonstrated that β-elemene effectively inhibited the growth and induced the apoptosis of Burkitt's lymphoma cells through upregulation of PUMA expression and modulating PUMA related apoptotic signaling pathway. The in vivo data confirmed the anti-tumor effect of β-elemene in the xenografts, suggesting that β-elemene is associated with PUMA activation, leading to Bax and caspase induction and onset of mitochondrial apoptosis., (Copyright © 2018 Elsevier Masson SAS. All rights reserved.)

Burkitt's lymphoma (BL) is a highly aggressive B-cell non-Hodgkin lymphoma with rapid growth and dissemination propensity. Triptolide (TP), an active component extracted from Chinese herb Tripterygium wilfordii Hook f., has broad-spectrum anti-tumor activities. This study aimed to explore the in vitro and in vivo anti-cancer effects of TP on BL and the potential molecular mechanisms. In this study, the in vitro anti-tumor activity of TP was determined by CCK-8 and flow cytometry assays in Raji, NAMALWA and Daudi cells. The expression of SIRT3, phosphorylation and acetylation of glycogen synthase kinase-3β (GSK-3β) were analyzed by Western blot assay. Moreover, we examined the mitochondrial membrane potential by JC-1 method and measured apoptosis related protein using Western blot assay. BL xenograft model in NOD/SCID mice were established to evaluate the in vivo anti-cancer effect of TP. We discovered that TP inhibited BL cell growth and induced apoptosis in a dose-dependent manner. Loss of SIRT3 provides growth advances for BL cells. However, TP could up-regulate SIRT3 expression, which resulted in suppression of BL cells proliferation. GSK-3β was activated by SIRT3-mediated deacetylation, which subsequently induced mitochondrial translocation and accumulation of Bax and decrease of mitochondrial membrane potential. Anti-tumor studies in vivo showed that TP (0.36 mg/kg) inhibited the growth of BL xenografts in NOD/SCID mice with an inhibitory rate of 73.13%. Our data revealed that TP triggered mitochondrial apoptotic pathway in BL by increasing SIRT3 expression and activating SIRT3/GSK-3β/Bax pathway. This study indicated that TP is a potential anti-cancer Chinese herbal medicine against BL., (Copyright © 2018 Elsevier Inc. All rights reserved.)

The aim of this project is to gain insights into the uptake and cellular actions of the enantiomeric R- and S-1,1'-binaphthyl-2,2'-diaminodichlorido-Pt(ii) complexes (R- and S-[Pt(DABN)Cl 2 ]) in the cisplatin-sensitive human Burkitt lymphoma cell line (Gumbus, IC 50 : 1.3 ± 0.2 μM) and its cisplatin-resistant sub-line (CDDPrGB, IC 50 : 6.6 ± 1.2 μM). The cellular uptakes of R- and S-[Pt(DABN)Cl 2 ] are ca. 4-fold higher than cisplatin, and involve a transport mechanism independent of the volume-sensitive, organic anion-channel complex, which facilitates cisplatin accumulation. The cisplatin-resistant CDDPrGB cells are not cross-resistant to either S- or R-[Pt(DABN)Cl 2 ]. We also find that even though R-[Pt(DABN)Cl 2 ] has a higher maximal cellular uptake and binds at higher levels to calf-thymus DNA than S-[Pt(DABN)Cl 2 ], it appears that S-[Pt(DABN)Cl 2 ] is more cytotoxic for Gumbus (IC 50 : 0.4 ± 0.1 μM) compared to R-[Pt(DABN)Cl 2 ] (IC 50 : 0.7 ± 0.3 μM). The cellular action of R- and S-[Pt(DABN)Cl 2 ] involves G0/G1 cell cycle arrest and cell death involving the extrinsic and intrinsic apoptotic pathways.

Myc plays important roles in cell cycle progression, cell growth, and stem cell self-renewal. Although dysregulation of Myc expression is a hallmark of human cancers, there is no Myc targeted therapy yet. Here, we report sAJM589, a novel small molecule Myc inhibitor, identified from a PCA-based high-throughput screen. sAJM589 potently disrupts the Myc-Max heterodimer in a dose dependent manner with an IC 50 of 1.8 ± 0.03 μM. sAJM589 preferentially inhibits transcription of Myc target genes in a Burkitt lymphoma cell model, P493-6. Genome-wide transcriptome analysis showed that sAJM589 treatment and Myc depletion induced similar gene expression profiles. Consistently, sAJM589 suppressed cellular proliferation in diverse Myc-dependent cancer cell lines and anchorage independent growth of Raji cells. Disruption of the Myc-Max interaction by sAJM589 reduced Myc protein levels, possibly by promoting ubiquitination and degradation of Myc. Collectively, these results suggest that sAJM589 may be a basis for the development of potential inhibitors of Myc-dependent cell growth.

Hsp90 is a molecular chaperone that protects proteins, including oncogenic signaling complexes, from proteolytic degradation. PU-H71 is a next-generation Hsp90 inhibitor that preferentially targets the functionally distinct pool of Hsp90 present in tumor cells. Tumors that are driven by the MYC oncoprotein may be particularly sensitive to PU-H71 due to the essential role of Hsp90 in the epichaperome, which maintains the malignant phenotype in the setting of MYC. Burkitt lymphoma (BL) is an aggressive B-cell lymphoma characterized by MYC dysregulation. In this study, we evaluated Hsp90 as a potential therapeutic target in BL. We found that primary BL tumors overexpress Hsp90 and that Hsp90 inhibition has antitumor activity in vitro and in vivo , including potent activity in a patient-derived xenograft model of BL. To evaluate the targets of PU-H71 in BL, we performed high-affinity capture followed by proteomic analysis using mass spectrometry. We found that Hsp90 inhibition targets multiple components of PI3K/AKT/mTOR signaling, highlighting the importance of this pathway in BL. Finally, we found that the anti-lymphoma activity of PU-H71 is synergistic with dual PI3K/mTOR inhibition in vitro and in vivo Overall, this work provides support for Hsp90 as a therapeutic target in BL and suggests the potential for combination therapy with PU-H71 and inhibitors of PI3K/mTOR. Mol Cancer Ther; 16(9); 1779-90. ©2017 AACR ., (©2017 American Association for Cancer Research.)

Inhibition of monocarboxylate transporter 1 has been proposed as a therapeutic approach to perturb lactate shuttling in tumor cells that lack monocarboxylate transporter 4. We examined the monocarboxylate transporter 1 inhibitor AZD3965, currently in phase I clinical studies, as a potential therapy for diffuse large B-cell lymphoma and Burkitt lymphoma. Whilst extensive monocarboxylate transporter 1 protein was found in 120 diffuse large B-cell lymphoma and 10 Burkitt lymphoma patients' tumors, monocarboxylate transporter 4 protein expression was undetectable in 73% of the diffuse large B-cell lymphoma samples and undetectable or negligible in each Burkitt lymphoma sample. AZD3965 treatment led to a rapid accumulation of intracellular lactate in a panel of lymphoma cell lines with low monocarboxylate transporter 4 protein expression and potently inhibited their proliferation. Metabolic changes induced by AZD3965 in lymphoma cells were consistent with a feedback inhibition of glycolysis. A profound cytostatic response was also observed in vivo : daily oral AZD3965 treatment for 24 days inhibited CA46 Burkitt lymphoma growth by 99%. Continuous exposure of CA46 cells to AZD3965 for 7 weeks in vitro resulted in a greater dependency upon oxidative phosphorylation. Combining AZD3965 with an inhibitor of mitochondrial complex I (central to oxidative phosphorylation) induced significant lymphoma cell death in vitro and reduced CA46 disease burden in vivo These data support clinical examination of AZD3965 in Burkitt lymphoma and diffuse large B-cell lymphoma patients with low tumor monocarboxylate transporter 4 expression and highlight the potential of combination strategies to optimally target the metabolic phenotype of tumors., (Copyright© 2017 Ferrata Storti Foundation.)

Silurus asotus egg lectin (SAL), an α-galactoside-binding protein isolated from the eggs of catfish, is a member of the rhamnose-binding lectin family that binds to Gb3 glycan (Galα1-4Galβ1-4Glc). We have previously demonstrated that SAL reduces the proliferation of Gb3-expressing Burkitt's lymphoma Raji cells and confirm here that it does not reduce their viability, indicating that unlike other lectins, it is not cytotoxic. The aim of this study was to determine the signal transduction mechanism(s) underlying this novel SAL/Gb3 binding-mediated effect profile. SAL/Gb3 interaction arrested the cell cycle through increasing the G 0/1 phase population of Raji cells. SAL suppressed the transcription of cell cycle-related factors such as c-MYC, cyclin D3, and cyclin-dependent protein kinase (CDK)-4. Conversely, the CDK inhibitors p21 and p27 were elevated by treatment with SAL. In particular, the production of p27 in response to SAL treatment increased steadily, whereas p21 production was maximal at 12 h and lower at 24 h. Activation of Ras-MEK-ERK pathway led to an increase in expression of p21. Notably, treatment of Raji cells with anti-Gb3 mAb alone did not produce the above effects. Taken together, our findings suggest that Gb3 on the Raji cell surface interacts with SAL to trigger sequential GDP-Ras phosphorylation, Ras-MEK-ERK pathway activation, p21 production, and cell cycle arrest at the G 0/1 phase.

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., (Copyright © 2016. Published by Elsevier Masson SAS.)

The effect of overexpression of Oct-1 protein isoforms on the cell response to two anticancer drugs camptothecin and dexamethasone was studied. The effect of Oct-1 isoforms on regulated gene transcription was estimated by the difference in the level of mRNA in Burkitt's lymphoma cells (Namalwa line) untransfected and stably transfected with Oct-1 isoforms. The response to anticancer drugs of the Oct-1 target genes involved in the development of apoptosis depended, firstly, on the type of drug, secondly, on the concentration of Oct-1 in cells. and, thirdly, on the Oct-1 isoform with which these cells were transfected.

Biotherapeutics are the fastest growing class of pharmaceutical with a rapidly evolving market facing the rise of biosimilar and biobetter products. In contrast to a biosimilar, which is derived from the same gene sequence as the innovator product, a biobetter has enhanced properties, such as enhanced efficacy or reduced immunogenicity. Little work has been carried out so far to increase the intrinsic stability of biotherapeutics via sequence changes, even though, aggregation, the primary degradation pathway of proteins, leads to issues ranging from manufacturing failure to immunological response and to loss of therapeutic activity. Using our spatial aggregation propensity tool as a first step to a rational design approach to identify aggregation-prone regions, biobetters of rituximab have been produced with enhanced stability by introducing site-specific mutations. Significant stabilization against aggregation was achieved for rituximab with no decrease in its binding affinity to the antigen., (© 2015 Wiley Periodicals, Inc. and the American Pharmacists Association.)

The present study demonstrates that fucose-containing sulfated polysaccharides (FCSP) from brown algae interfere with the CXCL12/CXCR4 axis in human Burkitt's lymphoma cells by binding CXCL12 and thereby blocking both CXCL12-induced CXCR4 receptor activation and downstream effects like migration and secretion of matrix metalloproteinase-9. This mode of action is currently considered as promising strategy for tumor therapy and may contribute to the known in vivo antitumor, antimetastatic and antiangiogenic activity of FCSP. In terms of the inhibition of the CXCR4 activation, FCSP from Saccharina latissima (S.l.-FCSP) proved to be more active than a commercial "Fucoidan" from Fucus vesiculosus, and both FCSP were superior to heparins by more than one order of magnitude. Fractionation of S.l.-FCSP revealed that its main fraction is composed of a homogeneous, higher sulfated galactofucan (S.l.-SGF) which consistently exhibited stronger activities and can therefore be considered as the active ingredient of S.l.-FCSP. By subjecting Fucoidan to the same fractionation procedure, the inhibitory activity of the obtained purified Fucoidan on the CXCL12/CXCR4 axis tended to be weaker and its antioxidant and antiproliferative effects were lost. This was probably due to the separation of contaminants including phenolic compounds, whose content additionally showed marked batch-to-batch variability. Regarding the need of standardized, well-characterized FCSP preparations for any potential medical application, our results indicate that S.l.-SGF is a promising candidate for further investigations and that S. latissima may be a more appropriate source of FCSP than F. vesiculosus or other algae species with high contents of co-extractable compounds., (© The Author 2015. Published by Oxford University Press. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

Up-regulation of glycolysis, a well recognized hallmark of cancer cells, was also found to be predictive of poor chemotherapy response. This observation suggested the attempt of sensitizing cancer cells to conventional chemotherapeutic agents by inhibiting glucose metabolism. Lactate dehydrogenase (LDH) inhibition can be a way to hinder glycolysis of cancer cells without affecting the metabolism of normal tissues, which usually does not require this enzymatic activity. In this paper, we showed that two LDH inhibitors (oxamate and galloflavin) can increase the efficacy of cisplatin in cultured Burkitt's lymphoma (BL) cells and that this potentiating effect is not exerted in proliferating normal lymphocytes. This result was explained by the finding that in BL cells LDH inhibition induced reactive oxygen species (ROS) generation, which was not evidenced in proliferating normal lymphocytes. In BL cells treated with the association of cisplatin and LDH inhibitors, these ROS can be a further cause of DNA damage, to be added to that produced by cisplatin, leading to the failure of the response repair. At present LDH inhibitors suitable for clinical use are actively searched; our results can allow a better understanding of the potentiality of LDH as a possible target to develop innovative anticancer treatments., (Copyright © 2015 Elsevier B.V. All rights reserved.)

Burkitt's lymphoma (BL) is a highly aggressive type of non-Hodgkin's lymphoma, with marked rates of proliferation and metabolism. The expression levels of the translocated cellular Myc (c-Myc) oncogene and Epstein-Barr virus infection have an oncogenic role in facilitating tumor progression and maintaining a malignant phenotype in BL Raji cells. The present study identified that more reactive oxygen species (ROS) were produced in Raji cells compared with other types of malignant B cells. Cells exhibiting higher ROS levels suggested facilitation of the induction of cell death by ROS-induction compounds. In the present study, apogossypolone (ApoG2) was observed to induce marked accumulation in the levels of ROS in the Raji cells, which damaged the cells and suppressed cell proliferation. Within 12 h following ApoG2 treatment, the Raji cells were prominently arrested in the G1 phase of the cell cycle. Immunoblotting analysis indicated that the chromodomain-helicase-DNA-binding protein 1, checkpoint kinase 1 and c-Myc proteins were significantly downregulated at 3, 6 and 12 h, respectively, following treatment. Following treatment with ApoG2 for 48 h, ApoG2 induced significant apoptosis in the Raji cells. This findings, together with our previous studies, which demonstrated ApoG2 as a potent inhibitor of anti-apoptotic B cell lymphoma 2 proteins, indicated that the ROS stimulatory effect of ApoG2 increased the antitumor activity of ApoG2.

We here report the synthesis of ethylene glycol N-interlinked imipramine dimers of various lengths from the tricyclic antidepressant desipramine via an amide coupling reaction followed by reduction with lithium aluminium hydride. The target molecules were found to be potent inhibitors of cellular viability while inducing cell type specific death mechanisms in three cancer cell lines including a highly chemoresistant Burkitt's lymphoma cell line. Basic amine analogues were found to be important for increased potency. Imipramine and desipramine were also tested for apoptotic activity and were found to be much less active than the novel dimeric compounds. Imipramine dimers were only found to be moderate inhibitors of the human serotonin transporter (hSERT) having IC(50) values in the micromolar region whilst the induction of cell death occurred independently of hSERT expression. These results demonstrate the potential of newly designed and synthesised imipramines derivatives for use against malignant cells, including those resistant to standard chemotherapy., (Copyright © 2013 Elsevier Ltd. All rights reserved.)

Piperlongumine (PL), a pepper plant alkaloid from Piper longum, kills solid tumor cells in a highly selective, potent fashion. To evaluate whether PL may have similar effects on malignant blood cells, we determined the efficacy with which PL inhibits the B-lymphocyte derived neoplasm, Burkitt lymphoma (BL). Low micromolar concentrations of PL (IC(50) = 2.8 μM × 8.5 μM) curbed growth and survival of two EBV(+) BL cell lines (Daudi, Raji) and two EBV BL cell lines (Ramos, DG-75), but left normal peripheral blood B-lymphocytes unharmed. PL-dependent cytotoxicity was effected in part by reduced NF-κB and MYC activity, with the former being caused by inhibition of IκBα degradation, nuclear translocation of p65, and binding of NF-κB dimers to cognate DNA sequences in gene promoters. In 4 of 4 BL cell lines, the NF-κB/MYC-regulated cellular target genes, E2F1 and MYB, were down regulated, while the stress sensor gene, GADD45B, was up regulated. The EBV-encoded oncogene, LMP-1, was suppressed in Daudi and Raji cells. Considering that NF-κB, MYC and LMP-1 play a crucial role in the biology of many blood cancers including BL, our results provide a strong preclinical rationale for considering PL in new intervention approaches for patients with hematologic malignancies., (Copyright © 2012 Elsevier Ltd. All rights reserved.)

Burkitt lymphoma (BL) is a highly aggressive B cell neoplasm. Although intensive polychemotherapy regimens have proven very effective, they are associated with significant toxicities. Therefore, more rational therapies that selectively target the molecular abnormalities of BL are needed. Recent data suggest that the tyrosine kinase SRC could represent a therapeutic target for BL. We found that new pyrazolo[3,4-d]pyrimidine SRC inhibitors exerted a significant cytotoxic effect and induced apoptosis on two BL cell lines, as determined by MTS assays, cytofluorimetric analyses and caspase 3 assay. Notably, our SRC inhibitors proved to be more effective than the well-known SRC inhibitor PP2 [4-amino-5-(4-chlorophenyl)-7-(dimethylethyl)pyrazolo[3,4-d]pyrimidine] in BL cells. Moreover, our small molecules induced a G 2/M arrest in BL cells through a possible new mechanism, whereby SRC inhibition hinders an AKT-WEE1-cyclin-dependent kinase 1 (CDK1) axis, leading to inhibition of CDK1, the main trigger of entry into mitosis. By using a small-molecule inhibitor of WEE1, a crucial CDK1 negative regulator, we were able to shift the balance toward apoptosis rather than growth arrest and enhance the efficacy of the SRC inhibitors, suggesting a possible use of these selective drugs in combination for a safe and efficient treatment of BL.

Plitidepsin (Aplidin), an antitumor agent of marine origin, presently is undergoing phase II/III clinical trials, and has shown promise for the treatment of lymphoma. Here, we describe the antitumor effects of plitidepsin alone and in combination with rituximab and investigated the effects of each drug and the combination on the cell cycle and mechanism of cell death. Several Diffuse Large Cell Lymphoma (DLCL) lines and Burkitt cell lines were tested for sensitivity to plitidepsin and rituximab. All DLCL and Burkitt lymphoma cell lines were inhibited by plitidepsin in nanomolar concentrations, while rituximab sensitivity varied among different cell lines. Ramos and the RL cell lines proved sensitive to rituximab and were used to test the effects of each of the two drugs. The two agents exhibited synergism at all tested concentrations. For in vivo studies, irradiated athymic nude mice were engrafted with the Ramos lymphoma. Treatment was initiated when the tumors were ~0.5 cm in diameter, and toxic and therapeutic effects were monitored. In the in vivo study, additive effects of the combined two drugs, was demonstrated without an increase in host toxicity. The in vitro synergy and the in vivo additive antitumor effects without an increase in host toxicity with two relatively non-marrow suppressive agents encourages further development of this combination for treatment of aggressive B-cell lymphomas.

A novel anticancer mechanism of catfish (Silurus asotus) egg lectin (SAL) was found to occur via the down-regulation of the membrane transopter protein, MRP1 (multidrug resistance associate protein-1) on Burkitt's lymphoma cells through Gb3(Galα1-4Galβ1-4Glc)-glycosphingolipid. Although SAL did not influence the viability of the cells directly, only 10 and 100 ng/mL of vincristine and etoposide, respectively induced anticancer effects when the lectin was applied in conjunction with these drugs. These phenomena were specifically inhibited by the co-presence of the α-galactoside, melibiose, which is a strong haptenic sugar of SAL that mimicks Gb3. The degree of expression regulation of the transporter proteins on the cells surface was investigated through the examination of the binding between SAL and Gb3-glycosphingolipid by immunological and molecular biological procedures. PCR data showed that MRP1 was more highly expressed when compared to another ATP-binding cassette family, multi-drug resistant protein and the expression levels of MRP1 on the cells were specifically dose- and time-dependently depleted by the addition of SAL. These results were also evaluated by immunological procedures using FACS and western-blotting. Small interfering RNA coding a part of MRP1 was transfected to Raji cells to knock down the protein, and cell death was increased by 10% when vincristine was administered at a concentration as low as 10 ng/mL compared to non-transfected cells. These results indicated that SAL possesses the potential to enhance the anticancer activites of low-concentrations of vincristine by the down-regulating the MRP1 gene expression to inhibit the multidrug resistance by binding to the target ligand Gb3-glycosphingolipid on Burkitt's lymphoma cells.

Epstein-Barr virus (EBV) is associated with a variety of lymphoid malignancies. Bortezomib activates EBV lytic gene expression. Bortezomib, a proteasome inhibitor, leads to increased levels of CCAAT/enhancer-binding proteinβ (C/EBPβ) in a variety of tumor cell lines. C/EBPβ activates the promoter of the EBV lytic switch gene ZTA. Bortezomib treatment leads to increased binding of C/EBP to previously recognized binding sites in the ZTA promoter. Knockdown of C/EBPβ inhibits bortezomib activation of EBV lytic gene expression. Bortezomib also induces the unfolded protein response (UPR), as evidenced by increases in ATF4, CHOP10, and XBP1s and cleavage of ATF6. Thapsigargin, an inducer of the UPR that does not interfere with proteasome function, also induces EBV lytic gene expression. The effects of thapsigargin on EBV lytic gene expression are also inhibited by C/EBPβ knock-down. Therefore, C/EBPβ mediates the activation of EBV lytic gene expression associated with bortezomib and another UPR inducer.

Previous studies have shown that 7-b (6-(dodecylamino)-2-(3-(4-methylpiperazin-1-yl)propyl)-1H-benzo-[de]isoquinoline-1,3(2H)-dione), a novel amonafide-based DNA intercalator, was generated as a new anticancer candidate. However, the effects induced by 7-b and the molecular mechanisms involved remain poorly understood in Burkitt's lymphoma. To shed light on these issues, we have investigated the effects of 7-b on proliferation, cell cycle progression, apoptosis activity and oxidative stress levels of lymphoma Raji cells in vitro. Our results showed that 7-b inhibited the proliferation of Raji cells and induced G1 cell cycle arrest in a dose-dependent manner. Moreover, 7-b treatment triggered programmed cell death, production of reactive oxygen species (ROS) and alteration of the mitochondrial membrane potential (Δψm). Altogether our results showed that 7-b mediated its growth inhibitory effects on Raji cells via the activation of a ROS-mediated mitochondrial pathway and cell cycle checkpoint signaling pathway which subsequently targeted p21., (Copyright © 2011 Elsevier Ltd. All rights reserved.)

Structural derivatives of 4-MTA, an illegal amphetamine analogue have been previously shown to have anticancer effects in vitro. In this study we report the synthesis of a series of novel 1,3-bis(aryl)-2-nitro-1-propene derivatives related in structure to 4-MTA. A number of these compounds containing a classic nitrostyrene structure are shown to have antiproliferative activities in vitro in a range of malignant cell lines, particularly against Burkitt's lymphoma derived cell lines, whilst having no effect on 'normal' peripheral blood mononuclear cells. Such effects appear to be independent of the serotonin transporter, a high affinity target for amphetamines and independent of protein tyrosine phosphatases and tubulin dynamics both of which have been previously associated with nitrostyrene-induced cell death. We demonstrate that a number of these compounds induce caspase activation, PARP cleavage, chromatin condensation and membrane blebbing in a Burkitt's lymphoma derived cell line, consistent with these compounds inducing apoptosis in vitro. Although no specific target has yet been identified for the action of these compounds, the cell death elicited is potent, selective and worthy of further investigation., (Copyright © 2010 Elsevier Ltd. All rights reserved.)

p53 protein induces cell cycle arrest, DNA repair, or apoptosis of damaged cells. Loss of wild-type p53 (wt-p53) function was shown to be associated with upregulation of survivin and resistance to therapy. Here we investigated the effects of DNA-damage agents in inducing apoptosis and cell cycle arrest, and modulation of survivin levels in two Burkitt's lymphoma (BL) cell lines with different p53 mutations. Our results showed that BL cell lines have variable response to DNA-damaging agents that cannot be correlated exclusively with p53 mutation or survivin expression suggesting that p53-independent transactivation may play a role in apoptosis induced by DNA-damaging agents.

The therapeutic monoclonal antibody rituximab has previously been shown to kill B cells in a caspase-independent manner. The signalling pathways underpinning this novel death pathway are unknown. The present study showed that rituximab treatment of Burkitt lymphoma cell lines induced a slow rise in intracellular calcium ([Ca(2+)](i)). This rise was only witnessed in cell lines that were killed by antibody, suggesting a critical role for Ca(2+) in mediating rituximab-driven caspase-independent cell death. Inhibition of the two main intracellular store-located Ca(2+) channels, i.e. the ryanodine and inositol-1,4,5-triphosphate receptor channels by dantrolene and xestospongen-c respectively did not prevent the rise in Ca(2+) seen with rituximab or protect cells from subsequent death. In sharp contrast, inhibition of Ca(2+) entry via plasma membrane channels with (2-aminoethoxy) diphenylborate or SKF-96365 or complete chelation of extracellular Ca(2+) with ethyleneglycol bis (aminoethylether) tetra-acetate inhibited the rise in [Ca(2+)](i) and increased cell viability. Together, these data suggest that ligation of the CD20 receptor with rituximab allows a slow sustained influx of Ca(2+) from the external environment that under certain conditions can lead to cell death.

Bortezomib, an inhibitor of the 26S proteasome, is currently approved for treatment of multiple myeloma and is being studied for therapy of non-Hodgkin's lymphoma. We found that Epstein-Barr virus (EBV)-positive B cells with type III latency were more susceptible to killing by bortezomib than those with type I latency. Bortezomib induced apoptosis of EBV lymphoblastoid cell lines (LCLs) by inducing cleavage of caspases 8 and 9; apoptosis was inhibited by pretreatment with a pan-caspase inhibitor. Bortezomib reduced the levels of the p50 and p65 components of the canonical NF-kappaB pathway and reduced the level of p52 in the noncanonical NF-kappaB pathway, which is induced by EBV LMP1. Bortezomib inhibited expression of cIAP-1, cIAP-2, and XIAP, which are regulated by NF-kappaB and function as inhibitors of apoptosis. Bortezomib did not inhibit expression of several other antiapoptotic proteins, including Bcl-2 and Bcl-XL. Finally, bortezomib significantly prolonged the survival of severe combined immunodeficiency mice inoculated with LCLs. These findings suggest that bortezomib may represent a novel strategy for the treatment of certain EBV-associated lymphomas.

Immunotherapy with rituximab (chimeric anti-CD20 monoclonal antibody, Rituxan), alone or in conjunction with chemotherapy, has significantly improved the treatment outcome of lymphoma patients. Via an elusive mechanism, a subpopulation of patients becomes unresponsive and/or relapses. To recapitulate various aspects of acquired resistance, rituximab-resistant (RR) clones were established from lymphoma lines and compared with parental cells. Surface CD20 expression was diminished in the clones. The clones neither responded to rituximab-mediated growth reduction or complement-dependent cytotoxicity nor underwent apoptosis in response to cross-linked rituximab. Rituximab failed to chemosensitize the RR clones, which exhibited constitutive hyperactivation of the nuclear factor-kappaB and extracellular signal-regulated kinase 1/2 pathways, leading to overexpression of B-cell lymphoma protein 2 (Bcl-2), Bcl-2-related gene (long alternatively spliced variant of Bcl-x gene), and myeloid cell differentiation 1 and higher drug resistance. Unlike parental cells, rituximab neither inhibited the activity of these pathways nor diminished the expression of resistant factors. Pharmacologic inhibitors of the survival pathways or Bcl-2 family members reduced the activity of these pathways, diminished antiapoptotic protein expression, and chemosensitized the RR clones. These novel in vitro results denote that continuous long-term rituximab exposure culminates in RR clones that do not respond to rituximab-mediated effects, have altered cellular signaling dynamics, and exhibit different genetic and phenotypic properties compared with parental cells. The data also reveal that although RR clones exhibit higher resistance to rituximab and cytotoxic drugs, these clones can be chemosensitized following treatment with pharmacologic inhibitors (e.g., dehydroxymethylepoxyquinomicin, bortezomib, PD098059) that target survival/antiapoptotic pathways. The findings also identify intracellular targets for potential molecular therapeutic intervention to increase treatment efficacy. The significance and potential clinical relevance of the findings are discussed.

Epigenetic silencing of regulatory genes by aberrant methylation contributes to tumorigenesis. DNA methyltransferase inhibitors (DNMTI) represent promising new drugs for anti-cancer therapies. The DNMTI 5-Azacytidine is effective against myelodysplastic syndrome, but induces switching of latent to lytic Epstein-Barr virus (EBV) in vitro and results in EBV DNA demethylation with the potential of induction of lytic EBV in vivo. This is of considerable concern given that recurrent lytic EBV has been linked with an increased incidence of EBV-associated lymphomas. Based on the distinct properties of action we hypothesized that the newer DNMTI Zebularine might differ from 5-Azacytidine in its potential to induce switching from latent to lytic EBV. Here we show that both 5-Azacytidine and Zebularine are able to induce expression of E-cadherin, a cellular gene frequently silenced by hypermethylation in cancers, and thus demonstrate that both DNMTI are active in our experimental setting consisting of EBV-harboring Burkitt's lymphoma Akata cells. Quantification of mRNA expression of EBV genes revealed that 5-Azacytidine induces switching from latent to lytic EBV and, in addition, that the immediate-early lytic infection progresses to early and late lytic infection. Furthermore, 5-Azacytidine induced upregulation of the latent EBV genes LMP2A, LMP2B, and EBNA2 in a similar fashion as observed following switching of latent to lytic EBV upon cross-linking of the B-cell receptor. In striking contrast, Zebularine did not exhibit any effect neither on lytic nor on latent EBV gene expression. Thus, Zebularine might be safer than 5-Azacytidine for the treatment of cancers in EBV carriers and could also be applied against EBV-harboring tumors, since it does not induce switching from latent to lytic EBV which may result in secondary EBV-associated malignancies.

SIRT1 and other NAD-dependent deacetylases have been implicated in control of cellular responses to stress and in tumorigenesis through deacetylation of important regulatory proteins, including p53 and the BCL6 oncoprotein. Hereby, we describe the identification of a compound we named cambinol that inhibits NAD-dependent deacetylase activity of human SIRT1 and SIRT2. Consistent with the role of SIRT1 in promoting cell survival during stress, inhibition of SIRT1 activity with cambinol during genotoxic stress leads to hyperacetylation of key stress response proteins and promotes cell cycle arrest. Treatment of BCL6-expressing Burkitt lymphoma cells with cambinol as a single agent induced apoptosis, which was accompanied by hyperacetylation of BCL6 and p53. Because acetylation inactivates BCL6 and has the opposite effect on the function of p53 and other checkpoint pathways, the antitumor activity of cambinol in Burkitt lymphoma cells may be accomplished through a combined effect of BCL6 inactivation and checkpoint activation. Cambinol was well tolerated in mice and inhibited growth of Burkitt lymphoma xenografts. Inhibitors of NAD-dependent deacetylases may constitute novel anticancer agents.

Peroxisome proliferator-activated receptor-gamma (PPARgamma), a member of the nuclear receptor superfamily, has been detected in several human leukemia cells. Recent studies reported that PPARgamma ligands inhibit cell proliferation and induce apoptosis in both normal and malignant B-lineage cells. We investigated the expression of PPARgamma and the effects of PPARgamma ligands on UTree-O2, Bay91 and 380, three B-cell acute lymphoblastic leukemia (B-ALL) cell lines with t(14;18), which show a poor prognosis, accompanying c-myc abnormality. Western blot analysis identified expression of PPARgamma protein and real-time PCR that of PPARgamma mRNA on the three cell lines. Troglitazone (TGZ), a synthetic PPARgamma ligand, inhibited cell growth in these cell lines in a dose-dependent manner, which was associated with G(1) cell cycle arrest and apoptosis. We also found this effect PPARgamma independent since PPARgamma antagonists failed to reverse this effect. We assessed the expression of c-myc, an apoptosis-regulatory gene, since c-myc abnormality was detected in most B-ALL cells with t(14;18). TGZ was found to dose-dependently downregulate the expression of c-myc mRNA and c-myc protein in the three cell lines. These results suggest that TGZ inhibits cell growth via induction of G(1) cell cycle arrest and apoptosis in these cell lines and that TGZ-induced apoptosis, at least in part, may be related to the downregulation of c-myc expression. Moreover, the downregulation of c-myc expression by TGZ may depend on a PPARgamma-independent mechanism. Further studies indicate that PPARgamma ligands may serve as a therapeutic agent in B-ALL with t(14;18).

Objectives: Members of the tumor necrosis factor family are potent inducers of apoptosis in sensitive cells and may be suitable for novel anti-cancer therapies aimed at inducing apoptosis via the activation of receptors with the death domain on malignant cells. We characterized the sensitivity of Burkitt's lymphoma (BL) cell lines to tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) and anti-Fas agonist, and investigated the mechanism of resistance of BL cell lines to TRAIL and Fas apoptotic pathways., Methods: Epstein-Barr virus (EBV) status in BL cell lines was determined by PCR. The extent of apoptosis following exposure to TRAIL and anti-Fas agonist was measured by 7A6 antigen staining. Expression of TRAIL receptors and Fas was determined by flow cytometry and reverse transcriptase-PCR. Western blot analyses were used to determine the expression of proapoptotic and antiapoptotic proteins. NF-kappaB activity was evaluated by electrophoretic mobility shift assay., Results: The sensitivity of BL cell lines to anti-Fas agonist depended on the expression of Fas. In contrast, the expression of TRAIL receptors did not correlate with the sensitivity to TRAIL-induced apoptosis. Interestingly, EBV-infected BL cell lines which showed constitutive levels of NF-kappaB activation, were TRAIL-resistant. NF-kappaB inhibitors reversed the resistance to TRAIL-induced apoptosis., Conclusions: Our results suggest that activation of NF-kappaB by EBV infection plays an important role in resistance of BL cell lines to TRAIL-induced apoptosis, and that NF-kappaB inhibitors may be useful adjuncts in clinical use of TRAIL against BL.

Ten patients developing lymphomas after disease modifying anti-rheumatic drugs (DMARD) (methotrexate, n = 3, mean cumulative dose = 3.4 g; cyclophosphamide, n = 2, mean dose = 70 g; azathioprine, n = 6, mean dose = 243 g) were investigated. Methotrexate-related lymphomas were Epstein-Barr virus (EBV)-positive, had infrequent aberrant methylation of p15 and p16, and responded well to methotrexate withdrawal or anti-CD20 antibody (rituximab) alone without concomitant chemotherapy, implying that defective immunosurveillance was important in lymphomagenesis. However, 75% of cyclophosphamide/azathioprine-related lymphomas were EBV-negative, had frequent p15 and p16 methylation, and responded poorly to drug withdrawal and chemotherapy, implying that direct drug-induced mutagenesis might be involved in lymphomagenesis., ((c) 2005 Wiley-Liss, Inc.)

The monoclonal antibody (mAb) rituximab produces objective clinical responses in patients with B-cell non-Hodgkin's lymphoma and antibody-based autoimmune diseases. Mechanisms mediating B-cell depletion by rituximab are not completely understood and may include direct effects of signalling via the target antigen CD20. Like most but not all CD20 mAbs, rituximab induces a sharp change in the solubility of the CD20 protein in the non-ionic detergent Triton-X-100, reflecting a dramatic increase in the innate affinity of CD20 for membrane raft signalling domains. Apoptosis induced by rituximab hypercrosslinking has been shown to require src family kinases (SFK), which are enriched in rafts. In this report we provide experimental evidence that SFK-dependent apoptotic signals induced by rituximab are raft dependent. Cholesterol depletion prevented the association of hypercrosslinked CD20 with detergent-insoluble rafts, and attenuated both calcium mobilization and apoptosis induced with rituximab. CD20 cocapped with the raft-associated transmembrane adaptor LAB/NTAL after hypercrosslinking with CD20 mAbs, regardless of their ability to induce a change in the affinity of CD20 for rafts. Taken together, the data demonstrate that CD20 hypercrosslinking via rituximab activates SFKs and downstream signalling events by clustering membrane rafts in which antibody-bound CD20 is localized in a high-affinity configuration.

In Burkitt's lymphoma (BL) cells c-myc is often translocated in proximity to the Emu enhancer of the Ig gene locus. This translocation causes c-myc hyperexpression and an increase in the cells' proliferative capacity. A peptide nucleic acid (PNA) complementary to enhancer Emu intronic sequence (PNAEmu), linked to a nuclear localization signal (NLS), selectively and specifically blocks the expression of the c-myc oncogene under Emu control in vitro, suggesting potential therapeutic use. To explore this issue further, we have determined the pharmacokinetics of (14)C-labeled PNAEmu in SCID mice where a human tumor is established by inoculation of cells from a BL cell line. The data demonstrate that the compound has a relatively long life in vivo in tissues and, in particular, in BL tumor mass. Furthermore, in this animal model, PNAEmu shows low or no toxicity. All these results are in favor of a successful preclinical application in a BL human tumor animal model of a PNA targeting a regulatory, nontranscribed DNA sequence that can selectively inhibit the hyperexpression of a translocated gene linked to neoplastic cell expansion.

The apoptotic pathway activated by chimeric anti-CD20 monoclonal antibodies (rituximab, IDEC.C2B8) was analyzed using the Burkitt lymphoma cell line Ramos. Crosslinking of CD20 (CD20XL) induced apoptosis in Ramos cells, which involved loss of mitochondrial membrane potential (Deltapsi(m)), the release of cytochrome-c (cyt-c), and activation of caspases-9 and -3. Nevertheless, several lines of evidence showed that the apoptotic outcome did not depend on these events. First, under circumstances where Ramos cells display resistance to either CD95- or B cell receptor (BCR)-induced apoptosis, CD20XL-induced apoptosis was not affected, pointing to a distinct pathway. Second, the broad-spectrum caspase inhibitor zVAD-fmk prevented processing of caspase-9, -3 and PARP as well as DNA fragmentation, but did not block apoptosis as measured by annexin V staining, cell size and membrane integrity. Lastly, Bcl-2 overexpression blocked cyt-c release and the decrease in Deltapsi(m), and completely prevented CD95- or BCR-mediated apoptosis; however, it did not affect CD20XL-induced cell death. We conclude that although CD20XL can initiate the mitochondrial apoptosis pathway, CD20-induced apoptosis does not necessarily require active caspases and cannot be blocked by Bcl-2. Since most chemotherapeutic drugs require the activation of caspases to exert their cytotoxicity, these findings provide an important rationale for the use of CD20 mAbs in chemoresistant malignancies.

The stilbene phytochemicals resveratrol and piceatannol have been reported to possess substantial antitumorigenic and antileukemic activities, respectively. Although recent experimental data revealed the proapoptotic potency of resveratrol, the molecular mechanisms underlying the antileukemic activity have not yet been studied in detail. In the present study, we show that resveratrol, as well as the hydroxylated analog piceatannol, are potent inducers of apoptotic cell death in BJAB Burkitt-like lymphoma cells with an ED50 concentration of 25 microM. Further experiments revealed that treatment of BJAB cells with both substances led to a concentration-dependent activation of caspase-3 and mitochondrial permeability transition. Using BJAB cells overexpressing a dominant-negative mutant of the Fas-associated death domain (FADD) adaptor protein to block death receptor-mediated apoptosis, we demonstrate that resveratrol- and piceatannol-induced cell death in these cells is independent of the CD95/Fas signaling pathway. To explore the antileukemic properties of both compounds in more detail, we extended our study to primary, leukemic lymphoblasts. Interestingly, piceatannol but not resveratrol is a very efficient inducer of apoptosis in this ex vivo assay with leukemic lymphoblasts of 21 patients suffering from childhood lymphoblastic leukemia (ALL).

Development of the chimeric mouse antihuman CD20 antibody, Rituximab, presented a notable advance in the treatment of patients with non-Hodgkin's lymphoma (NHL). Its use allowed the specific targeting of tumor B cells without the systemic toxicity of traditional therapies. The mechanisms by which Rituximab induces its antitumor activity are not fully understood. We have shown previously that Rituximab down-regulates Bcl-2 expression in some B-NHL cell lymphoma lines through an interleukin 10 (IL-10)-dependent autocrine loop, an effect that renders the resistant cells susceptible to chemotherapeutic drugs. The objective of this study was to delineate the signaling pathway by which Bcl-2 is controlled by Rituximab and IL-10. We hypothesized that the down-regulation of IL-10 by Rituximab decreases activation of the signal transducer and activator of transcription 3 (STAT3) protein, which in turn, is responsible for decreased levels of Bcl-2. We demonstrate by phosphoprotein immunoblotting and gel shift analyses that endogenous IL-10 induces activation of STAT3 in the 2F7 cell line. Furthermore, we show that Rituximab and anti-IL-10 antibody treatment decreases the ability of STAT3 to bind to its DNA binding site. The decrease in STAT3 activation by these treatments correlates with a decrease in Bcl-2 expression. Additionally, piceatannol, an inhibitor of STAT3 activation, down-regulates the expression of Bcl-2. Altogether, these results demonstrate that Bcl-2 expression is under the regulation of the STAT3 signaling pathway, which is regulated by endogenously secreted IL-10. Hence, Rituximab-induced down-regulation of IL-10 expression is responsible for the down-regulation of Bcl-2 and sensitization of NHL cells by therapeutic drugs. Furthermore, these findings support the notion that circulating IL-10 in vivo may control the resistance of NHL to drug-mediated cytotoxicity.

Burkitt's lymphoma cell lines have been important in vitro models for studying the pathogenesis of Burkitt's lymphoma (BL) and for exploring new treatment strategies. A new EBV(-) Burkitt's lymphoma cell line (GA-10) was established from a patient with a clinically aggressive, chemorefractory BL and characterized. Although functional p-glycoprotein could not be demonstrated by dye-efflux assays, both p53 genes were mutated in the GA-10 cells, perhaps contributing to the resistant phenotype of the original neoplasm. Two properties of BL cells which may be useful targets for novel cytotoxic therapeutics are their surface expression of CD77, the receptor for Shiga toxin (Stx), and their high rate of proliferation. Expression of CD77 on the GA-10 cells was heterogeneous in that certain subclones expressed high levels of CD77 and correspondingly exhibited strong growth inhibition by Stx while others showed low levels of CD77 expression and weak Stx-induced growth inhibition. Flavopiridol, a potent inhibitor of cell cycle progression through G1 and G2, induced cytotoxicity of the GA-10 cells with an LC(50) of approximately 40 nM vs 70 nM for HL-60 cells (P < 0.05). The concentrations of flavopiridol at which only 10% of the cells were viable (LC(10)) were approximately 280 nM for the GA-10 cells and 520 nM for the HL-60 cells (P < 0.05). Dose-related induction of apoptosis in response to flavopiridol was demonstrated in the GA-10 cells by morphology, TUNEL assay, and activation of caspase-3. Flavopiridol was also cytotoxic to seven other BL cell lines tested. These data suggest that flavopiridol may have therapeutic value in the treatment of Burkitt's lymphoma., (Copyright 2001 Academic Press.)

Administration of doxorubicin (DXR) formulated in sterically stabilized liposomes, (SL) containing engrafted poly(ethylene glycol)-modified phosphatidylethanolamine (PEG-PE) on their surface, has been shown to increase the therapeutic index of the drug. A further improvement could be achieved through targeting of liposome-entrapped drug selectively to cancer cells. This paper describes the conjugation of the anti-B-cell lymphoma monoclonal antibody LL2 to the surface of DXR-loaded liposomes by use of a PEG-based heterobifunctional coupling agent. Competitive-binding ELISA of the resulting immunoliposomes (SIL) against the monoclonal anti-idiotype antibody, WN, indicated preserved immunological activity. The pH-sensitive probe, HPTS was used to study the binding of liposomes with target cells. The results showed a 3.8-fold increased cellular association of SIL compared to that of SL and an apparent internalization of SIL into low pH compartments. Addition of an excess of unconjugated free LL2 displaced about 72% of the HPTS-SIL association with cells. Experiments with 125I-labeled free and SIL-bound LL2 showed approximately 50% degradation for both preparations. In vitro MTT cytotoxicity tests against neoplastic B cells gave IC(50) values of 1.6, 2.9 and 0.35 microM for DXR-SIL, DXR-SL and free DXR, respectively. Leakage of drug from the liposomes apparently reduced the specificity of the cytotoxic action of DXR-SIL.

We have previously demonstrated that 13-cis-retinoic acid (RA), 9-cis-RA and all-trans-RA (ATRA) powerfully inhibit the proliferation of Epstein-Barr virus-immortalized B-lymphoblastoid cell lines (LCLs). The aim of the present study was to assess whether these compounds are effective at inhibiting the growth of B cells at more advanced stages of lymphomagenesis, including fully transformed B lymphocytes. To this end, c-myc-transfected LCLs (myc-LCLs) and Burkitt's lymphoma (BL) cell lines were used. We report that 13-cis-RA, 9-cis-RA and ATRA also markedly inhibit the proliferation of myc-LCLs by inducing G(0)/G(1) growth arrest as well as enhancing rates of apoptosis. Conversely, all but 1 (DG75) of the 8 BL cell lines investigated were poorly RA-responsive. Moreover, unlike LCLs and myc-LCLs, RA-treated DG75 cells rapidly resumed proliferation upon drug removal. Analysis of cell cycle-regulatory proteins showed that, as in LCLs, strong up-regulation of p27(Kip-1) and increased levels of under-phosphorylated pRb and p130 were detected in RA-treated DG75 cells. While the catalytic activity of all 3 G(1)-associated CDKs (CDK2, CDK4 and CDK6) was strongly inhibited in RA-treated LCLs, only CDK2-associated kinase activity was reduced in DG75 cells arrested in G(0)/G(1) by RA. Moreover, RA-treated DG75 cells failed to show the down-regulation of cyclin D3 observed in LCLs. Use of receptor-selective agonists and antagonists showed that in LCLs and RA-responsive BL cells, RA-induced growth arrest is mainly mediated by RARalpha. The RARalpha-selective agonist Ro 40-6055 was also effective at very low concentrations (10(-10) M). Nevertheless, comparable levels of RARalpha mRNA were found in RA-responsive and -resistant BL cell lines, indicating that mechanisms different from transcriptional deregulation of RARalpha probably underlie the differential responsiveness of BL cells., (Copyright 2000 Wiley-Liss, Inc.)

Purpose: Methotrexate (MTX) remains one of the most effective drugs for the treatment of children with acute lymphoblastic leukemia (ALL). Because MTX and 5-methyltetrahydrofolate (5CH3THF) share uptake and metabolic pathways, the efficacy of MTX is likely to depend not only on its metabolism but also on how well folate is accumulated by lymphoblasts. The authors' goal was to compare in vitro folate and antifolate uptake in B-lineage lymphoblasts from patients who remained in continuous complete remission (CCR) and those in whom relapse occurred., Patients and Methods: Twenty-four children with B-lineage ALL were studied at diagnosis (n = 20) or relapse (n = 4). Lymphoblasts obtained by bone marrow aspiration were incubated for 24 hours in vitro with 0.05 microM 5CH3[3H]THF or 1 microM [3H]MTX., Results: As of July 1999, 16 patients studied at diagnosis remained in CCR at a median follow-up of 45 months after achieving remission. Two of the patients studied at relapse are in second CCR; the remaining two died from progressive disease. The median uptake of neither [3H]MTX nor 5CH3[3H]THF differed significantly between the 16 patients in first CCR studied at diagnosis and the 4 patients studied at relapse. However, the median ratio of [3H]MTX:5CH3[3H]THF uptake differed significantly for patients who remained in first CCR versus patients studied at relapse., Conclusions: The uptake of [3H]MTX in relation to 5CH3[3H]THF by leukemic lymphoblasts in vitro may correlate positively with treatment outcome in children with B-lineage ALL. A larger study of homogeneously treated patients is necessary to confirm these results.

The ratio of Bax to Bcl-2 protein can determine whether cells will die via apoptosis or be protected from it. Reh was found to express a high basal level of Bcl-2 but was lacking of Bax protein expression. Treatment with bryostatin 1 induced a down-regulation in Bcl-2 protein that was not accompanied by an obvious Bax protein induction or apoptosis. These results suggest that a decreased level of Bcl-2 alone in this cell line is not sufficient for apoptosis induction. In an effort to identify the mechanism whereby apoptosis could be induced in this ALL model, we treated Reh cells with three microtubule inhibitors: dolastatin 10, auristatin PE and vincristine, in the presence and absence of bryostatin 1. When used alone, only dolastatin 10 induced apoptosis that was detected morphologically, and by flow cytometry. Western blots revealed that dolastatin 10-induced apoptosis was accompanied by the induction of Bax protein and the reduction in Bcl-2 protein. Auristatin PE and vincristine induced both Bax and Bcl-2 protein, leaving the Bax:Bcl-2 ratio constant. Reh cells pretreated for 24 h with bryostatin 1 followed by dolastatin 10, auristatin PE or vincristine showed significant apoptosis which was accompanied by Bcl-2 protein down regulation and Bax protein up regulation. We conclude that: (1) expression of bax is necessary for apoptosis-induction in this model; (2) a decrease in Bcl-2 level alone is not sufficient and might not be necessary for apoptosis-induction; and (3) the ratio of Bax:Bcl-2 plays a critical role in susceptibility to apoptosis in Reh cells. The results from this study should prove useful in guiding the clinical application of these novel agents in the treatment of acute lymphoblastic leukemia.