Anti-Proliferative and Pro-Apoptotic vLMW Fucoidan Formulas Decrease PD-L1 Surface Expression in EBV Latency III and DLBCL Tumoral B-Cells by Decreasing Actin Network.

Epstein-Barr virus (EBV) infects 95% of the world's population and persists latently in the body. It immortalizes B-cells and is associated with lymphomas. LCLs (lymphoblastoid cell lines, EBV latency III B-cells) inhibit anti-tumoral T-cell response following PD-L1 overexpression (programmed death-ligand 1 immune checkpoint). Many cancer cells, including some DLBCLs (diffuse large B-cell lymphomas), also overexpress PD-L1. Immunotherapies are based on inhibition of PD-L1/PD-1 interactions but present some dose-dependent toxicities. We aim to find new strategies to improve their efficiency by decreasing PD-L1 expression. Fucoidan, a polysaccharide extracted from brown seaweed, exhibits immunomodulatory and anti-tumor activities depending on its polymerization degree, but data are scarce on lymphoma cells or immune checkpoints. LCLs and DLBCLs cells were treated with native fucoidan (Fucus vesiculosus) or original very-low-molecular-weight fucoidan formulas (vLMW-F). We observed cell proliferation decrease and apoptosis induction increase with vLMW-F and no toxicity on normal B- and T-cells. We highlighted a decrease in transcriptional and PD-L1 surface expression, even more efficient for vLMW than native fucoidan. This can be explained by actin network alteration, suggesting lower fusion of secretory vesicles carrying PD-L1 with the plasma membrane. We propose vLMW-F as potential adjuvants to immunotherapy due to their anti-proliferative and proapoptotic effects and ability to decrease PD-L1 membrane expression.

EBV Latency III-Transformed B Cells Are Inducers of Conventional and Unconventional Regulatory T Cells in a PD-L1-Dependent Manner.

EBV infects and immortalizes B cells in vitro and in vivo. It is the causative agent of most immune deficiency-related lymphoproliferative disorders and is associated with various lymphomas. EBV latency III-transformed B cells are known to express two immunosuppressive molecules, IL-10 and PD-L1, two characteristics of regulatory B cells (Bregs). In this study, we show that, in addition to secretion of the Breg immunosuppressive cytokines IL-10, IL-35, and TGF-ß1, EBV latency III-transformed B cells were able to repress proliferation of their autologous T cells preactivated by CD2, CD3, and CD28. This inhibitory effect was likely caused by CD4+ T cells because EBV latency III-transformed B cells induced a strong proliferation of isolated autologous CD8 T cells. Indeed, EBV was able to promote expansion of autologous FOXP3+ CD39high CTLA4+, Helios+, GITR+, LAG3+ CD4 T cells (i.e., regulatory T cells [Tregs]). Two types of Tregs were induced: unconventional CD25neg and conventional CD25pos Tregs. These Tregs expressed both the latency-associated peptide (LAP) and the PD-1 receptor, two markers of functional Tregs. Expansion of both Treg subtypes depended on PD-L1, whose expression was under the control of LMP1, the main EBV oncogene. These results demonstrate that, like Bregs, EBV latency III-transformed B cells exhibit strong immunoregulatory properties. These data provide clues to the understanding of how after EBV primo-infection, EBV-proliferating B cells can survive in an aggressive immunological environment and later emerge to give rise to EBV-associated B cell lymphomas such as in elderly patients.

Design and Synthesis of Tubulin and Histone Deacetylase Inhibitor Based on iso-Combretastatin A-4.

Designing multitarget drugs have raised considerable interest due to their advantages in the treatment of complex diseases such as cancer. Their design constitutes a challenge in antitumor drug discovery. The present study reports a dual inhibition of tubulin polymerization and HDAC activity. On the basis of 1,1-diarylethylenes ( isoCA-4) and belinostat, a series of hybrid molecules was successfully designed and synthesized. In particular compounds, 5f and 5h were proven to be potent inhibitors of both tubulin polymerization and HDAC8 leading to excellent antiproliferative activity.

B7-H1, which represses EBV-immortalized B cell killing by autologous T and NK cells, is oppositely regulated by c-Myc and EBV latency III program at both mRNA and secretory lysosome levels.

EBV-immortalized B cells induce a complex immune response such that the virus persists as a clinically silent infection for the lifetime of the infected host. B7-H1, also called PD-L1, is a cosignaling molecule of the B7 family that can inhibit activated T cell effectors by interaction with its receptor PD-1. In this work, we have studied the dependence of B7-H1 on NF-κB and c-Myc, the two main transcription factors in EBV latency III proliferating B cells, on various lymphoblastoid and Burkitt lymphoma cell lines, some of them being inducible or not for the EBV latency III program and/or for c-Myc. We found that B7-H1 repressed killing of EBV-immortalized B cells by their autologous T and NK cells. At the mRNA level, NF-κB was a weak inducer whereas c-Myc was a strong repressor of B7-H1 expression, an effect mediated by STAT1 inhibition. At the protein level, B7-H1 molecules were stored in both degradative and unconventional secretory lysosomes. Surface membrane B7-H1 molecules were constitutively internalized and proteolyzed in lysosomes. The EBV latency III program increased the amounts of B7-H1-containing secretory lysosomes and their export to the surface membrane. By repressing actin polymerization, c-Myc blocked secretory lysosome migration and B7-H1 surface membrane export. In addition to B7-H1, various immunoregulatory molecules participating in the immunological synapse are stored in secretory lysosomes. By playing on actin polymerization, c-Myc could thus globally regulate the immunogenicity of transformed B cells, acting on export of secretory lysosomes to plasma membrane.

Glycotranscriptome study reveals an enzymatic switch modulating glycosaminoglycan synthesis during B-cell development and activation.

B-cell fate and responses are modulated by soluble mediators and direct cellular interactions. Migration properties also vary during differentiation, commitment and activation. In many cells, modulation of responses to stimuli involves cell surface glycans, whose architecture depends on the simultaneous expression of multiple enzymes. By looking at the glycosylation-related gene expression patterns among B-cell populations, we determined in this study that the strongest variations were observed for CSGalNAcT-1 and EXTL1. These are enzymes involved in the biosynthesis of alternative forms of glycosaminoglycans (GAGs), namely chondroitin sulfate and heparan sulfate, respectively. These two enzymes showed inverse fluctuations in progenitors, resting B cells and activated B cells, suggesting a developmentally regulated switch between chondroitin and heparan sulfate synthesis. To explore whether these variations contributed to optimal B-cell differentiation, we overexpressed EXTL1 in the B-cell lineage of transgenic mice, yielding a partial differentiation blockade at the pro-B to pre-B transition. In the periphery, this defect was almost fully compensated for in vivo, with normal-size B-cell compartments and normal serum immunoglobulin levels in the transgenic EXTL1 mice. The peripheral B cells from EXTL1 transgenics were only affected with regard to their in vitro responses to polyclonal activation, showing reduced proliferation. Together the data suggest that despite their low amounts in lymphocytes, the heparan sulfate chains decorating the endogenous GAGs appear to be regulators of B-cell physiology.

Molecular basis of cytotoxicity of Epstein-Barr virus (EBV) latent membrane protein 1 (LMP1) in EBV latency III B cells: LMP1 induces type II ligand-independent autoactivation of CD95/Fas with caspase 8-mediated apoptosis.

The Epstein-Barr virus (EBV) oncoprotein latent membrane protein 1 (LMP1) is thought to act as the major transforming protein in various cell types, by rerouting the tumor necrosis factor receptor family signaling pathway. Despite this implication in EBV-associated transformation of cells, LMP1 toxicity is a well-known but poorly studied feature, perhaps because it contradicts its role in transformation. We show that LMP1 physiological levels are very heterogeneous and that the highest levels of LMP1 correlate with Fas overexpression and spontaneous apoptosis in lymphoblastoid cell lines (LCLs). To understand the cytotoxic effect of LMP1 in LCLs, we cloned wild-type LMP1 into a doxycycline double-inducible episomal vector pRT-1, with a truncated version of NGFR as a surrogate marker of inducibility. We found that LMP1 overexpression induced apoptosis in LCL B cells, as shown by annexin V labeling, sub-G(1) peak, and poly(ADP ribose) polymerase cleavage. Knocking down Fas expression by small interfering RNA abolished LMP1-induced apoptosis. The absence of detectable levels of Fas ligand mRNA suggested a ligand-independent activation of Fas. LMP1 induced Fas overexpression with its relocalization in lipid raft microdomains of the membrane. Fas immunoprecipitation detected FADD (Fas-associated death domain protein) and caspase 8, suggesting a Fas-dependent formation of the death-inducing signaling complex. Caspases 8, 9, 3, and 7 were activated by LMP1. Caspase 8 activation was associated with BID cleavage and truncated-BID mitochondrial relocalization, consistent with type II apoptosis. Therefore, our results are in agreement with a model where LMP1-dependent NF-kappaB activation induces Fas overexpression and autoactivation that could overwhelm the antiapoptotic effect of NF-kappaB, revealing an ambivalent function of LMP1 in cell survival and programmed cell death.

Differential involvement of mitochondria during ursolic acid-induced apoptotic process in HaCaT and M4Beu cells.

Ursolic acid (UA) is a pentacyclic triterpenoid compound which exists widely in nature and is known to have a pleitropic biological activity profile. For the last few decades, extensive work has been carried out to establish its biological activities and pharmacological actions. It is described as a promising chemopreventive agent with an antiproliferative effect on cancer cells that stems from its ability to induce apoptosis. We investigated and compared the role played by mitochondria during the apoptotic process induced by UA in human HaCaT-derived keratinotic cells and M4Beu human melanoma cells. In both cell lines, UA induced significant caspase-3 activation, the downstream central effector of apoptosis. Subsequent JC-1/TOTO-3 double staining clearly demonstrated that UA induces strong mitochondrial-transmembrane potential collapse in M4Beu cells, while mitochondria from HaCaT-treated cells remain largely unstimulated. This was confirmed by Western blot analysis, which revealed a Bax/Bcl-2-balance change in favor of Bax, the proapoptotic member, in UA-treated M4Beu cells. It can be concluded that UA induces apoptosis in M4Beu through the mitochondrial pathway, while other mechanisms are activated in the case of HaCaT cells.

Mitochondrial localization and activity of P-glycoprotein in doxorubicin-resistant K562 cells.

It is now well-established that P-glycoprotein 170 (P-gp), an efflux pump involved in multidrug resistance (MDR) is overexpressed at the plasma membrane of doxorubicin-resistant K562 leukemia cells. Nevertheless, several results suggested: (i) that P-gp-mediated drug efflux was not the only mechanism involved in resistance; (ii) that intracellular compartments could accumulate the drug, preventing it from reaching its nuclear targets; (iii) that agents able to reverse multidrug resistance may lead to intracellular drug redistribution. We have studied the localization of P-gp in mitochondria as well as its functional properties in this compartment. Using several monoclonal antibodies (MoAbs) directed against different P-gp epitopes, a protein was detected in the cytoplasm of two doxorubicin-resistant K562 sublines and, by confocal laser scanning microscopy, this protein was shown to co-localize in the Golgi apparatus and in mitochondria, in equivalent proportions. Purified mitochondria were isolated from K562 cell variants; the presence of a protein of about 170 kDa and reacting with several anti-P-gp antibodies was assessed in MDR cells by Western blotting and flow cytometry. Functional assays have shown that mitochondrial P-gp was involved in doxorubicin accumulation inside the organelle but not in its efflux, suggesting an orientation of P-gp in the mitochondrial membrane inverse to that observed in the plasma membrane. A potential role for mitochondrial P-gp in MDR cells would be to protect the nucleus from doxorubicin. This is the first demonstration of the presence and functional activity of P-gp in mitochondria of MDR cells.

Ursolic acid induces apoptosis through caspase-3 activation and cell cycle arrest in HaCat cells.

Ursolic acid (UA) is a pentacyclic triterpene compound isolated from many kinds of medicinal plants and present in human diet. In this study, we investigated the pro-apoptotic effect of UA on HaCat derived keratinocyte cell line. Treatment with UA decreased the viability of HaCat cells in a concentration- and time-dependent manner. In addition, cell cycle analysis revealed that UA treated HaCat cells were blocked predominantly in G1 phase. Moreover, expression of p21WAF1, a cell cycle regulator, was increased by UA, indicating that UA-induced cell cycle arrest could be mediated through p21WAF1. During UA treatment, we also demonstrated that p53 was phosphorylated at serine 392 and translocated to the nucleus. It is well established that p53 achieves its tumor suppressor activity by inducing apoptosis on cells. To define the apoptotic process in our system, we examined effect of UA on caspase activities, and demonstrated caspase-3 activation. In conclusion, our results suggest that UA induces: i) cell cycle arrest concomitantly with the apparition of the apoptotic sub group G1 peak, and ii) cell death through apoptosis, which is mediated by caspase-3.