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Rocaglates are natural compounds that have been extensively studied for their ability to inhibit translation initiation. Rocaglates represent promising drug candidates for tumor treatment due to their growth‐inhibitory effects on neoplastic cells. In contrast to natural rocaglates, synthetic analogues of rocaglates have been less comprehensively characterized, but were also shown to have similar effects on the process of protein translation. Here, we demonstrate an enhanced growth‐inhibitory effect of synthetic rocaglates when combined with glucose anti‐metabolite 2‐deoxy‐D‐glucose (2DG) in different cancer cell lines. Moreover, we unravel a new aspect in the mechanism of action of synthetic rocaglates involving reduction of glucose uptake mediated by downregulation or abrogation of glucose transporter GLUT‐1 expression. Importantly, cells with genetically induced resistance to synthetic rocaglates showed substantially less pronounced treatment effect on glucose metabolism and did not demonstrate GLUT‐1 downregulation, pointing at the crucial role of this mechanism for the anti‐tumor activity of the synthetic rocaglates. Transcriptome profiling revealed glycolysis as one of the major pathways differentially regulated in sensitive and resistant cells. Analysis of synthetic rocaglate efficacy in a 3D tissue context with a co‐culture of tumor and normal cells demonstrated a selective effect on tumor cells and substantiated the mechanistic observations obtained in cancer cell lines. Increased glucose uptake and metabolism is a universal feature across different tumor types. Therefore, targeting this feature by synthetic rocaglates could represent a promising direction for exploitation of rocaglates in novel anti‐tumor therapies., We demonstrate that novel synthetic rocaglate compounds induce cancer cell death through inhibition of glucose metabolism and glucose uptake mediated by downregulation of glucose transporter‐1 (GLUT‐1). Generation of a rocaglate‐resistant haploid cell line revealed the importance of GLUT‐1 downregulation as a major target for synthetic rocaglates and resistance development. Additionally, the combination with the glucose anti‐metabolite 2DG enhances the anti‐neoplastic activity of synthetic rocaglates, laying the foundation for novel promising rocaglate‐based combination therapy approaches.

Chronic lymphocytic leukemia is a malignancy of mature B cells that strongly depend on microenvironmental factors, and their deprivatio n has been ide nt ified as a promising treatmen t approach for this incurable disease. Cytokine array screening of 247 chronic lymphocytic leukemia serum samples revealed elevated levels of tumor necrosis factor (TNF) receptor-1 which were associated with poor clinical outcome. We detected a microenvironment-induced expression of TNF receptor-1 in chronic lymphocytic leukemia cells in vitro, and an aberrantly high expression of this receptor in the proliferation centers of patients’ lymph nodes. Stimulation of TNF receptor-1 with TNF-α enhanced nuclear factor κ-light-chain-enhancer of activated B cells (NFκB) activity and viability of chronic lymphocytic leukemia cells, which was inhibited by wogonin. The therapeutic effects of wogonin were analyzed in mice after adoptive transfer of Em-T-cell leukemia 1 (TCL1) leukemic cells. Wogonin treatment prevented leukemia development when given early after transplantation. The treatment of fullblown leukemia resulted in the loss of the TNF receptor-1 on chronic lymphocytic leukemia cells and their mobilization to blood. Targeting TNF receptor-1 signaling is therefore proposed for the treatment of chronic lymphocytic leukemia.

Multiple myeloma (MM) is an incurable malignancy by the presently known therapies. TRAIL is a promising anticancer agent that virtually not shows any toxicity to normal cells. We have recently carried out clinical trials with a human circularly permuted TRAIL, CPT, against MM saw a partial response in approximate 20–30% of patients. In the current study, we investigated the cause of CPT resistance and revealed that the majority of the MM patients express elevated levels of c-FLIP. Knockdown of c-FLIP expression by siRNA alone was sufficient to increase CPT-mediated apoptosis in a CPT-resistant human MM cell line U266. To overcome CPT resistance, we investigated the combination of CPT with Rocaglamides(s) in MM which has been shown to inhibit c-FLIP expression in vitro. We show that Rocaglamide(s) overcomes CPT resistance in U266 in vitro and significant increases in anti-tumor efficacies of CPT in mice xenografted with U266. Similar results were also obtained in mice xenografted with the CPT-resistant human acute T-cell leukemia cell line Molt-4. Our study suggests that the combination of Rocaglamide(s) with CPT may provide a more efficient treatment against myeloma and leukemia.

Over-expression of Bcl-2, Bcl-xL and Bcl-w is frequently associated with cancer resistance to chemotherapy. Navitoclax (ABT-263), an orally bio-available small-molecule mimetic of the Bcl-2 homology domain 3, specifically inhibits Bcl-2, Bcl-xL and Bcl-w. Despite promising results obtained from the clinical trials, the use of Navitoclax in patients is dose-limited due to induction of death of platelets via inhibition of Bcl-xL and subsequent thrombocytopenia. This side effect limits the use of Navitoclax in low doses and to very sensitive tumors. In this study, we show that HTLV-1-associated adult T-cell leukemia/lymphoma (ATL) cells, which over-express Bcl-2, Bcl-xL and Bcl-w, show a 10- to 20-fold higher sensitivity (EC50 = ∼ 25-50 nM) to Navitoclax compared to non-HTLV-1-associated leukemic cells (EC50 = ∼ 1 μM). Investigation of the molecular mechanisms revealed that the HTLV-1 oncogenic protein Tax up-regulates expression of the pro-apoptotic protein Bax which enhances the therapeutic efficacy of Navitoclax. In addition, we show that agents that inhibit the transcription elongation or translation initiation such as Wogonin and Roc-A can further decrease the effective dose of Navitoclax. Our study suggests that HTLV-1 ATL may be a good candidate disease for low dose Navitoclax therapy and probably with less risk of thrombocytopenia.

Natural compounds are an important source for drug development. With an increasing cancer rate worldwide there is an urgent quest for new anti‐cancer drugs. In this study, we show that a group of dolabrane‐type of diterpenes, collectively named tagalsins, isolated from the Chinese mangrove genus Ceriops has potent cytotoxicity on a panel of hematologic cancer cells. Investigation of the molecular mechanisms by which tagalsins kill malignant cells revealed that it induces a ROS‐mediated damage of DNA. This event leads to apoptosis induction and blockage of cell cycle progression at S‐G2 phase via activation of the ATM/ATR—Chk1/Chk2 check point pathway. We further show that tagalsins suppress growth of human T‐cell leukemia xenografts in vivo. Tagalsins show only minor toxicity on healthy cells and are well tolerated by mice. Our study shows a therapeutic potential of tagalsins for the treatment of hematologic malignancies and a new source of anticancer drugs., What's new? Mangroves of genus Ceriops, widespread and highly utilized in China, are of growing interest in anticancer drug development due to their production of potentially cytotoxic diterpenoids and triterpenoids. Here, a group dolabrane‐type diterpenes known as tagalsins isolated from the species C. tagal are shown to possess potent killing effects on cancer cells of hematologic origin. Cell death was associated with the production of reactive oxygen species and DNA damage. In vivo, tagalsins significantly delayed the development of human T‐cell leukemia in a murine xenograft model.

Rocaglamides (= flavaglines) are potent natural anti-cancer phytochemicals that inhibit cancer growth at nanomolar concentrations by the following mechanisms: (1) inhibition of translation initiation via inhibition of phosphorylation of the mRNA cap-binding eukaryotic translation initiation factor eIF4E and stabilization of RNA-binding of the translation initiation factor eIF4A in the eIF4F complex; (2) blocking cell cycle progression by activation of the ATM/ATR-Chk1/Chk2 checkpoint pathway; (3) inactivation of the heat shock factor 1 (HSF1) leading to up-regulation of thioredoxin-interacting protein (TXNIP) and consequent reduction of glucose uptake and (4) induction of apoptosis through activation of the MAPK p38 and JNK and inhibition of the Ras-CRaf-MEK-ERK signaling pathway. Besides the anti-cancer activities, rocaglamides are also shown to protect primary cells from chemotherapy-induced cell death and alleviate inflammation- and drug-induced injury in neuronal tissues. This review will focus on the recently discovered molecular mechanisms of the actions of rocaglamides and highlights the benefits of using rocaglamides in cancer treatment.

Targeting the cancer cell cycle machinery is an important strategy for cancer treatment. Cdc25A is an essential regulator of cycle progression and checkpoint response. Over-expression of Cdc25A occurs often in human cancers. In this study, we show that Rocaglamide-A (Roc-A), a natural anticancer compound isolated from the medicinal plant Aglaia, induces a rapid phosphorylation of Cdc25A and its subsequent degradation and, thereby, blocks cell cycle progression of tumor cells at the G1-S phase. Roc-A has previously been shown to inhibit tumor proliferation by blocking protein synthesis. In this study, we demonstrate that besides the translation inhibition Roc-A can induce a rapid degradation of Cdc25A by activation of the ATM/ATR-Chk1/Chk2 checkpoint pathway. However, Roc-A has no influence on cell cycle progression in proliferating normal T lymphocytes. Investigation of the molecular basis of tumor selectivity of Roc-A by a time-resolved microarray analysis of leukemic vs. proliferating normal T lymphocytes revealed that Roc-A activates different sets of genes in tumor cells compared with normal cells. In particular, Roc-A selectively stimulates a set of genes responsive to DNA replication stress in leukemic but not in normal T lymphocytes. These findings further support the development of Rocaglamide for antitumor therapy.

The role of Wilms' tumor suppressor 1 (WT1) in leukemogenesis has been investigated mostly in acute (AML) and chronic (CML) myeloid leukemias. So far, its oncogenic role has been controversially discussed because both overexpression and inactivating mutations are found. A recent study on primary samples from patients with acute T-cell leukemia (T-ALL) revealed that most of them do not express WT1 proteins although they express WT1 mRNA. In our study, we investigated WT-1 expression in ten T-ALL cell lines established from leukemia/lymphoma patients. We show that consistent with the finding in primary T-ALL cells, most of the leukemic T-cell lines tested do not overexpress WT1 proteins. We found that leukemic T-cells overexpressing WT1 protein produce higher levels of CD95L and show elevated CD95L-mediated activation-induced cell death (AICD) compared to cells lacking or expressing low levels of WT1. Ectopic expression of WT1 in the WT1-nonexpressing leukemic T-cell line increases CD95L expression and elevates activation-induced apoptosis, whereas silencing WT1 expression in the WT1-overexpressing leukemic T-cell line by siRNA confers reduced CD95L expression and reduction in AICD. Chromatin immunoprecipitation and luciferase-promoter reporter analysis demonstrate that WT1 binds to and enhances CD95L promoter activity through the Egr-binding sites. Our study provides a new role of WT1 in regulation of CD95L-mediated cell death.

Hodgkin/Reed-Sternberg lymphoma (HL) is a clonal B-cell-related malignancy. Although many patients with HL can be cured by the current regimen of high-dose multi-agent chemotherapy, the treatment causes high risks of later pathologies including secondary malignancies. This fact highlights the demand to develop rational treatment for HL. Survival and growth of HL cells are largely dependent on their microenvironment. In this study, using the HL cell lines L1236 and KM-H2 as model systems, we investigated the role of IL-4/IL-13 signaling in regulation of drug sensitivity and resistance in HL. We show that specific blocking of IL-4 and IL-13-mediated STAT6 activation by either an IL-4-binding fusion protein APG598 or an IL-4R antagonist APG201 (R121D/Y124D) renders HL cells more prone to apoptotic killing by chemotherapeutic drugs such as Mitomycin C, 5-Fluorouracil, Etopside, Doxorubicin and Paclitaxel. This effect is due to inhibition of STAT6-mediated elevation of expression of the anti-apoptotic Bcl-2 family protein Bcl-xL. Employing ChIP analysis in combination with APG201 or STAT6-specific siRNA we identified a defined STAT6-binding site in the Bcl-xL promoter region from -1967 to -1957 of the transcription start site. Our data demonstrate that the IL-4/IL-13-STAT6-Bcl-xL axis may be an important target for HL treatment. This study also suggests that combination of classical chemotherapeutic drugs with the IL-4/IL-13 antagonists may enhance efficacy and reduce risks of toxicity from high dose of drugs in HL treatment.

Curcumin is the active ingredient of the spice turmeric and has been shown to have a number of pharmacologic and therapeutic activities including antioxidant, anti-microbial, anti-inflammatory, and anti-carcinogenic properties. The anti-inflammatory effects of curcumin have primarily been attributed to its inhibitory effect on NF-κB activity due to redox regulation. In this study, we show that curcumin is an immunosuppressive phytochemical that blocks T cell-activation-induced Ca(2+) mobilization with IC(50) = ∼12.5 μM and thereby prevents NFAT activation and NFAT-regulated cytokine expression. This finding provides a new mechanism for curcumin-mediated anti-inflammatory and immunosuppressive function. We also show that curcumin can synergize with CsA to enhance immunosuppressive activity because of different inhibitory mechanisms. Furthermore, because Ca(2+) is also the secondary messenger crucial for the TCR-induced NF-κB signaling pathway, our finding also provides another mechanism by which curcumin suppresses NF-κB activation.

Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) is a promising anticancer agent that kills various tumor cells without damaging normal tissues. However, many cancers remain resistant to TRAIL. To overcome TRAIL resistance, combination therapies using sensitizers of the TRAIL pathway would be an efficacious approach. To investigate potential sensitizers of TRAIL-induced apoptosis, we used TRAIL-resistant human T cell leukemia virus type 1 (HTLV-1)-associated adult T cell leukemia/lymphoma (ATL) cells as a model system. So far, HTLV-1-associated ATL is incurable by presently known therapies. Here, we show that wogonin and the structurally related natural flavones apigenin and chrysin break TRAIL resistance in HTLV-1-associated ATL by transcriptional down-regulation of c-FLIP, a key inhibitor of death receptor signaling, and by up-regulation of TRAIL receptor 2 (TRAIL-R2). This effect is mediated through transcriptional inhibition of the p53 antagonist murine double minute 2 (Mdm2), leading to an increase in p53 levels and, consequently, to up-regulation of the p53 target gene TRAIL-R2. We also show that these flavones can sensitize to TNFα- and CD95-mediated cell death. Furthermore, we show that wogonin, apigenin, and chrysin also enhance TRAIL-mediated apoptosis in other human cancer cell lines including breast cancer cell line MDA-MB-231, colon cancer cell line HT-29, hepatocellular carcinoma cell line HepG2, melanoma cell line SK-MEL-37, and pancreatic carcinoma cell line Capan-1 by the same mechanism. Thus, our study suggests the potential use of these flavones as an adjuvant for TRAIL-mediated anticancer therapy.

The early growth response gene product Egr-1 has been shown to have great impact on growth, proliferation, and differentiation in a wide variety of cells, including T cells. In this study, we show that Egr-1 is rapidly induced upon T cell stimulation and is expressed predominantly in T helper type 2 (Th2) compared with type 1 (Th1) cells. We further investigate the role of Egr-1 in regulation of the Th2 cytokine interleukin-4 (IL-4) expression. IL-4 is a key Th2 cytokine that regulates humoral immunity and also causes allergic inflammation. Regulation of IL-4 gene transcription in Th2 cells has been shown to be controlled by multiple T cell receptor (TCR)-induced transcription factors. However, only a few transcription factors were shown to be selectively induced in differentiated Th2 cells in response to TCR stimulation. Chromatin immunoprecipitation analysis demonstrates that Egr-1 binds to the IL-4 promoter in vivo upon T cell stimulation. Ectopic expression of Egr-1 enhances endogenous IL-4 mRNA expression and elevates IL-4 promoter activity. We also show that Egr-1, nuclear factor of activated T cell, and NF-kappaB cooperatively bind to an NFAT/NF-kappaB-overlapping IL-4 enhancer element and activate the IL-4 promoter synergistically. Furthermore, we show that antisense oligonucleotides that knock down Egr-1 expression attenuate IL-4 transcription. Our study provides the first evidence that Egr-1 protein is differentially expressed in Th1 and Th2 cells and is involved in the acute phase of the IL-4 transcription in response to TCR stimulation.

Drugs with tumor selectivity may have an important benefit in chemotherapies. We have previously shown that Rocaglamide(s), derived from the medicinal plant Aglaia, kills various leukemic cells through the mitochondrial apoptosis pathway with only minor toxicities to normal lymphocytes. Here, we show further that Rocaglamide preferentially promotes activation-induced cell death in malignant T cells by differential regulation of c-FLIP and CD95L expression. Rocaglamide enhances and also prolongs activation-induced JNK activation in malignant T cells leading to downregulation of c-FLIP but upregulation of CD95L expression. We also show that malignant T cells express a significantly higher amount of Bid - the molecular linker that bridges the receptor-mediated to the mitochondria-mediated apoptosis pathway. Conversely, a substantially lower amount of c-FLIP in response to T-cell stimulation compared to normal T cells is observed. This difference may provide a therapeutic window for cancer treatment. The effect of Rocaglamide on sensitization of activation-induced cell death in malignant T cells was further demonstrated in vivo in a mouse model. Our study demonstrates that Rocaglamide may be a potential anticancer drug that simultaneously targets both c-FLIP and CD95L expressions in tumor cells. This study may also provide a new clue to design a more efficient chemotherapy by using a combination of stimuli that engage the receptor-mediated and the mitochondria-mediated death pathway.

With an increasing cancer rate worldwide, there is an urgent quest for the improvement of anticancer drugs. One of the main problems of present chemotherapy in treatment of tumor patients is the toxicity of drugs. Most of the existent anticancer drugs, unfortunately, attack also proliferating normal cells. In recent years, traditional Chinese herbal remedies have gradually gained considerable attention as a new source of anticancer drugs. Although their healing mechanisms are still largely unknown, some of the drugs have been used to help cancer patients fight their disease at reduced side effects compared to other treatments. In our study, we show that Rocaglamide (Roc), derived from the traditional Chinese medicinal plants Aglaia, induces apoptosis through the intrinsic death pathway in various human leukemia cell lines and in acute lymphoblastic leukemia, chronic myeloid leukemia and acute myeloid leukemia cells freshly isolated from patients. Investigation of the molecular mechanisms by which Roc kills tumors revealed that it induces a consistent activation of the stress-response mitogen-activated protein kinase (MAPK) p38 accompanied with a long-term suppression of the survival MAPK extracellular signal-regulated kinase. These events affect proapoptotic Bcl-2 family proteins leading to depolarization of the mitochondrial membrane potential and trigger caspase-mediated apoptosis involving caspase-9, -8, -3 and -2. Importantly, Roc shows no effects on MAPKs in normal lymphocytes and therefore has no or very low toxicity on healthy cells. Up to now, more than 50 different Roc derivatives have been isolated from Aglaia. Our study suggests that Roc derivatives may be promising candidates for the development of new drugs against hematologic malignancies.

Chemical examination of the barks of mangrove plant Ceriops tagal resulted in the isolation of six new dolabrane-type diterpenes with the trivial names of tagalenes A-F (1-6), together with 10 known analogues. The structures of new compounds were elucidated by extensive spectroscopic data analyses. Tagalenes A-C (1-3) are characterized by 18-nordolabrane scaffold, while tagalene F (6) featured by a 2,3-seco dolabrane derivative. Antitumor assay revealed that seven compounds exhibited potent inhibitory effects against a panel of tumor cell lines with IC50

TNFα has previously been used in anticancer therapy. However, the therapeutic application of TNFα was largely limited due to its general toxicity and the fact that it activates the NF-κB–family transcription factors, which are proinflammatory and antiapoptotic. To overcome this problem in vitro, specific NF-κB inhibitors or transcription or protein synthesis inhibitors such as actinomycin D and cycloheximide are usually used in combination to increase TNFα killing of tumor cells. However, these agents also cause harmful side effects in vivo. We show here that wogonin, derived from the popular Chinese herb Huang-Qin, attenuates NF-κB activity by shifting TNFα-induced free radical ·O2– to a more reduced nonradical product, H2O2, and thereby sensitizes TNFα-resistant leukemia cells to TNFα-induced apoptosis. Importantly, wogonin does not affect the viability of normal peripheral blood T cells. Wogonin also sensitizes TRAIL-induced apoptosis. Our data suggest a potential use of wogonin as a TNFα or TRAIL adjuvant for cancer treatment. Our data also demonstrate how a herbal compound enhances killing of tumor cells with reduced side effects compared with other treatments.

The HTLV-1 transactivator protein Tax is essential for malignant transformation of CD4 T cells, ultimately leading to adult T-cell leukemia/lymphoma (ATL). Malignant transformation may involve development of apoptosis resistance. In this study we investigated the molecular mechanisms by which HTLV-1 Tax confers resistance toward CD95-mediated apoptosis. We show that Tax-expressing T-cell lines derived from HTLV-1–infected patients express elevated levels of c-FLIPL and c-FLIPS. The levels of c-FLIP correlated with resistance toward CD95-mediated apoptosis. Using an inducible system we demonstrated that both resistance toward CD95-mediated apoptosis and induction of c-FLIP are dependent on Tax. In addition, analysis of early cleavage of the BH3-only Bcl-2 family member Bid, a direct caspase-8 substrate, revealed that apoptosis is inhibited at a CD95 death receptor proximal level in Tax-expressing cells. Finally, using siRNA we directly showed that c-FLIP confers Tax-mediated resistance toward CD95-mediated apoptosis. In conclusion, our data suggest an important mechanism by which expression of HTLV-1 Tax may lead to immune escape of infected T cells and, thus, to persistent infection and transformation.

Over-expression of Bcl-2, Bcl-xL and Bcl-w is frequently associated with cancer resistance to chemotherapy. Navitoclax (ABT-263), an orally bio-available small-molecule mimetic of the Bcl-2 homology domain 3, specifically inhibits Bcl-2, Bcl-xL and Bcl-w. Despite promising results obtained from the clinical trials, the use of Navitoclax in patients is dose-limited due to induction of death of platelets via inhibition of Bcl-xL and subsequent thrombocytopenia. This side effect limits the use of Navitoclax in low doses and to very sensitive tumors. In this study, we show that HTLV-1-associated adult T-cell leukemia/lymphoma (ATL) cells, which over-express Bcl-2, Bcl-xL and Bcl-w, show a 10- to 20-fold higher sensitivity (EC50 = ∼ 25-50 nM) to Navitoclax compared to non-HTLV-1-associated leukemic cells (EC50 = ∼ 1 μM). Investigation of the molecular mechanisms revealed that the HTLV-1 oncogenic protein Tax up-regulates expression of the pro-apoptotic protein Bax which enhances the therapeutic efficacy of Navitoclax. In addition, we show that agents that inhibit the transcription elongation or translation initiation such as Wogonin and Roc-A can further decrease the effective dose of Navitoclax. Our study suggests that HTLV-1 ATL may be a good candidate disease for low dose Navitoclax therapy and probably with less risk of thrombocytopenia.

Endothelial cells are the primary targets of circulating immune and inflammatory mediators. We hypothesize that interleukin-18, a proinflammatory cytokine, induces endothelial cell apoptosis. Human cardiac microvascular endothelial cells (HCMEC) were treated with interleukin (IL) 18. mRNA expression was analyzed by ribonuclease protection assay, protein levels by immunoblotting, and cell death by enzyme-linked immunosorbent assay and fluorescence-activated cell sorter analysis. We also investigated the signal transduction pathways involved in IL-18-mediated cell death. Treatment of HCMEC with IL-18 increases 1) NF-kappaB DNA binding activity; 2) induces kappaB-driven luciferase activity; 3) induces IL-1beta and TNF-alpha expression via NF-kappaB activation; 4) inhibits antiapoptotic Bcl-2 and Bcl-X(L); 5) up-regulates proapoptotic Fas, Fas-L, and Bcl-X(S) expression; 6) induces fas and Fas-L promoter activities via NF-kappaB activation; 7) activates caspases-8, -3, -9, and BID; 8) induces cytochrome c release into the cytoplasm; 9) inhibits FLIP; and 10) induces HCME cell death by apoptosis as seen by increased annexin V staining and increased levels of mono- and oligonucleosomal fragmented DNA. Whereas overexpression of Bcl-2 significantly attenuated IL-18-induced endothelial cell apoptosis, Bcl-2/Bcl-X(L) chimeric phosphorothioated 2'-MOE-modified antisense oligonucleotides potentiated the proapoptotic effects of IL-18. Furthermore, caspase-8, IKK-alpha, and NF-kappaB p65 knockdown or dominant negative IkappaB-alpha and dominant negative IkappaB-beta or kinase dead IKK-beta significantly attenuated IL-18-induced HCME cell death. Effects of IL-18 on cell death are direct and are not mediated by intermediaries such as IL-1beta, tumor necrosis factor-alpha, or interferon-gamma. Taken together, our results indicate that IL-18 activates both intrinsic and extrinsic proapoptotic signaling pathways, induces endothelial cell death, and thereby may play a role in myocardial inflammation and injury.

IL-4 plays a pivotal role in the development of the Th2 cell mediated humoral immune response and causes IgE-dependent allergic inflammatory diseases. Expression of IL-4 in differentiated Th2 cells is regulated by transcription factors such as NF-AT, AP-1 and NF-IL6. Recently, increasing evidence indicates that the pro-inflammatory transcription factor NF-kappa B may also participate inIL-4 expression. In this study, we show that the IL-4 promoter is synergistically activated by NF-kappa B, NF-AT and NF-IL6 at the NF-kappa B/NF-AT/NF-IL6 composite sites. In addition, we performed the chromatin immunoprecipitation technique to determine the functional relevance of NF-kappa B in the activation of the IL-4 gene in vivo. We demonstrate that NF-kappa B binds to the IL-4 promoter in vivo upon T cell activation. Inhibition of NF-kappa B nuclear translocation in living cells blocked binding of NF-kappa B to the IL-4 promoter. The data provide first evidence that NF-kappa B is directly involved in IL-4 transcription.

CD95/CD95L mediated apoptosis is an important mechanism of immune homeostasis. It is instrumental for termination of an immune response and mainly be involved in peripheral tolerance. Dysregulation of the CD95/CD95L system leads to severe diseases. In this review, we present a survey of the role of the CD95/CD95L system in the immune system and, particularly, focus on the signals and transcription factors (NF-AT, Egr, NF-kappaB, AP-1, c-Myc, Nur77, IRFs, SP-1, ALG-4, ROR(gamma)t, and CIITA) involved in CD95L expression. It should also be evident from this review that a profound insight into the molecular mechanisms of CD95L activation should allow to explore potential therapeutic means to treat CD95/CD95L-dependent diseases.

Sesquiterpene lactones (SL) derived from Mexican India medicinal plants and parthenolide, the major SL from European feverfew, have raised considerable interest because of their anti-inflammatory and complex pharmacological action. Interleukin-4 (IL-4) is a key cytokine that influences the development of T helper 2 cells and plays an important role in the pathogenesis of allergic diseases. We show here that the anti-inflammatory parthenolide suppresses IL-4 expression at the mRNA and the protein levels in a dose-dependent manner. We demonstrate that parthenolide blocks NF-kappaB binding to two important IL-4 promoter regulatory elements and suppresses promoter activity upon T cell activation. Differences regarding the effects of parthenolide on expression levels of IL-4, IL-2 and IFN-gamma were observed. Parthenolide (2.5 microM) could completely suppress IL-4 protein levels secreted in anti-CD3/CD28-stimulated peripheral blood T cells from allergic and normal donors. Complete inhibition of IL-2 and IFN-gamma requires higher doses of parthenolide. So far, drugs directed against IL-4 expression have not been described. This finding raises the potential to develop parthenolide to treat IL-4-mediated allergic-like inflammation.

Polarization of CD4(+) T helper cells toward either a Th1 or Th2 response can significantly influence host immunity to pathogens. IL-4 and IFN-gamma are the signature cytokines of Th2 and Th1 cells, respectively. IFN-gamma was shown to assist Th1 development by promoting IL-12 and IL-12 receptor expression. So far, direct influence of Th2 cytokine expression by IFN-gamma has not been described. We show here that IFN-gamma directly suppresses IL-4 gene expression. IRF-1 and IRF-2 induced by IFN-gamma bind to three distinct IL-4 promoter sites and function as transcriptional repressors. Our data demonstrate a direct negative feedback of IFN-gamma on expression of the Th2 cytokine gene IL-4 and, thus, provide evidence for another important mechanism by which IFNgamma assists Th1 and attenuates Th2 responses.

Apoptosis is a morphologically distinct form of cell death involved in many physiological and pathological processes. The death receptor CD95 (APO-1/Fas) and its ligand (L) CD95L are critically involved in activation-induced-cell-death (AICD) of activated T-cells. Here we show that the anti-inflammatory sesquiterpene lactone parthenolide derived from the European traditional herb-medicine feverfew and many Mexican India medicinal plants suppresses expression of the CD95L and CD95 at the mRNA levels, thus, preventing T-cells from AICD. We demonstrate that parthenolide blocks NF-kappaB binding to the two NF-kappa binding sites of the CD95L promoter and suppresses promoter activity upon T-cell activation. Aberrant expression of CD95 and, particularly CD95L is dangerous and may lead to severe diseases. Our study indicates that parthenolide supports T-cell survival by down-regulating the CD95 system, at least in part, and, therefore, may have therapeutic potential as a new anti-apoptotic substance against AICD in T-cells.

IL-4 plays a pivotal role in the development of allergic inflammation via induction of IgE isotype switching, increase of IgE receptor expression, promoting Th2 cell differentiation, and stimulating several genes involved in atopic disorders. Previous studies in human and mouse models have shown that high vitamin E intake correlates with low IgE concentration and reduced prevalence of allergic reactions. We, therefore, investigated the mechanism of the vitamin E effect on T cells. We show here that the natural free radical scavenger vitamin E suppresses IL-4 protein levels in humanperipheral blood T cells in a dose-dependent manner. The effect of vitamin E on IL-4 expression occurs at the mRNA level. Vitamin E has been implicated in inhibition of DNA binding and function of the redox-regulated transcription factors NF-κB and AP-1. Investigation of the molecular mechanism by which vitamin E suppresses IL-4 transcription shows that it blocks binding of transcription factors to two important IL-4 promoter binding sites for NF-κB and AP-1 and interferes with promoter activity upon T cell activation. Our data provide molecular evidence supporting the beneficial effect of dietary vitamin E on atopic disorders.

Apoptosis is a morphologically distinct form of cell death involved in many physiological and pathological processes. Expression of the CD95 (APO-1/Fas) ligand (CD95L) is critically involved in activation-induced cell death (AICD) of activated T cells. Here we show that the natural free radical scavenger vitamin E suppresses the activity of the transcription factors NF-kappa B and AP-1, thus blocking expression of CD95L and preventing T cell AICD. Since AICD is a major cause of T cell depletion in AIDS, we examined 35 HIV-1-positive individuals and found that their T cells are more susceptible to AICD than are T cells isolated from healthy controls. Administration of vitamin E suppresses CD95L mRNA expression and protects T cells of HIV-1-infected individuals from CD95-mediated apoptosis. This evidence that vitamin E can affect T cell survival may merit further clinical investigation.

The CD95 (also called APO-1/Fas) system plays a major role in the induction of apoptosis in lymphoid and nonlymphoid tissues. The CD95 ligand (CD95L) is induced in response to a variety of signals, including IFN-γ and TCR/CD3 stimulation. Here we report the identification of two positive regulatory IFN-regulatory factor-dependent domains (PRIDDs) in the CD95L promoter and its 5′ untranslated region, respectively. EMSAs demonstrate specific binding of IFN-γ-induced IFN-regulatory factor 1 (IRF-1) to the PRIDD sequences. Ectopic IRF-1 expression induces CD95L promoter activity. Furthermore, we demonstrate that PRIDDs play an important role in TCR/CD3-mediated CD95L induction. Most interestingly, viral IRFs of human herpes virus 8 (HHV8) totally abolish IRF-1-mediated and strongly reduce TCR/CD3-mediated CD95L induction. We demonstrate here for the first time that viral IRFs inhibit activation-induced cell death. Thus, these results demonstrate an important mechanism of HHV8 to modulate the immune response by down-regulation of CD95L expression. Inhibition of CD95-dependent T cell function might contribute to the immune escape of HHV8.

Tumor initiation, progression and resistance to therapies are tightly associated with over-expression of anti-apoptotic proteins Bcl-2, Bcl-x(L), Bcl-w and Mcl-1. ABT-263 (Navitoclax), an orally bio-available small-molecule mimetic of the Bcl-2 homology domain 3, inhibits Bcl-2, Bcl-x(L), and Bcl-w and has shown anti-cancer effects mainly on lymphomas and lymphocytic leukemia. Despite promising results obtained from the clinical trials, the use of ABT-263 in patients is dose-limited due to causing thrombocytopenia via inhibition of Bcl-x(L) in platelets. ABT-199 specifically inhibits Bcl-2; however, its use is limited to tumors over-expressing only Bcl-2. Besides, many tumors resist treatment due to high levels of Mcl-1 expression or develop resistance via up-regulation of Mcl-1 during long-term exposure. These obstacles highlight the demand to improve the ABT-263-based therapy. In this study, we show that anti-cancer flavones, e.g., wogonin, baicalein, apigenin, chrysin and luteolin enhance ABT-263-induced apoptosis in different cancer cell lines and in primary AML and ALL cells by down-regulation of Mcl-1 expression. Importantly, wogonin does not enhance the toxicity of ABT-263 to proliferating normal T cells and thrombocytes. Wogonin also potentiates the lethality of ABT-263 in cancer cells which have acquired resistance to ABT-263. Furthermore, we show that combination of wogonin with ABT-263 promotes in vivo tumor regression in a human T-cell leukemia xenograft mouse model. Our study demonstrates that wogonin (and related flavones) reduce the effective dose of ABT-263 thereby possibly decreasing the risk of adverse side effects.

Tumor initiation, progression and resistance to therapies are tightly associated with over-expression of anti-apoptotic proteins Bcl-2, Bcl-x(L), Bcl-w and Mcl-1. ABT-263 (Navitoclax), an orally bio-available small-molecule mimetic of the Bcl-2 homology domain 3, inhibits Bcl-2, Bcl-x(L), and Bcl-w and has shown anti-cancer effects mainly on lymphomas and lymphocytic leukemia. Despite promising results obtained from the clinical trials, the use of ABT-263 in patients is dose-limited due to causing thrombocytopenia via inhibition of Bcl-x(L) in platelets. ABT-199 specifically inhibits Bcl-2; however, its use is limited to tumors over-expressing only Bcl-2. Besides, many tumors resist treatment due to high levels of Mcl-1 expression or develop resistance via up-regulation of Mcl-1 during long-term exposure. These obstacles highlight the demand to improve the ABT-263-based therapy. In this study, we show that anti-cancer flavones, e.g., wogonin, baicalein, apigenin, chrysin and luteolin enhance ABT-263-induced apoptosis in different cancer cell lines and in primary AML and ALL cells by down-regulation of Mcl-1 expression. Importantly, wogonin does not enhance the toxicity of ABT-263 to proliferating normal T cells and thrombocytes. Wogonin also potentiates the lethality of ABT-263 in cancer cells which have acquired resistance to ABT-263. Furthermore, we show that combination of wogonin with ABT-263 promotes in vivo tumor regression in a human T-cell leukemia xenograft mouse model. Our study demonstrates that wogonin (and related flavones) reduce the effective dose of ABT-263 thereby possibly decreasing the risk of adverse side effects.

One of the main obstacles of conventional anticancer therapy is the toxicity of chemotherapeutics to normal tissues. So far, clinical approaches that aim to specifically reduce chemotherapy-mediated toxicities are rare. Recently, a number of studies have demonstrated that herbal extracts derived from traditional Chinese medicine (TCM) may reduce chemotherapy-induced side effects. Thus, we screened a panel of published cancer-inhibiting TCM compounds for their chemoprotective potential and identified the phytochemical Rocaglamide (Roc-A) as a candidate. We show that Roc-A significantly reduces apoptotic cell death induced by DNA-damaging anticancer drugs in primary human and murine cells. Investigation of the molecular mechanism of Roc-A-mediated protection revealed that Roc-A specifically blocks DNA damage-induced upregulation of the transcription factor p53 by inhibiting its protein synthesis. The essential role of p53 in Roc-A-mediated protection was confirmed by siRNA knockdown of p53 and by comparison of the effects of Roc-A on chemoprotection of splenocytes isolated from wild-type and p53-deficient mice. Importantly, Roc-A did not protect p53-deficient or -mutated cancer cells. Our data suggest that Roc-A may be used as an adjuvant to reduce the side effects of chemotherapy in patients with p53-deficient or -mutated tumors.

In HIV-infected individuals dysregulation of the immune system is characterized by severe disorders of the cytokine network. Increase secretion of IL-2, the major T cell growth and differentiation cytokine, may play a decisive role in sensitization of T cells for activation induced apoptosis and indirect death of activated T cells through augmented virus replication. We investigated the cause of enhanced IL-2 secretion and found that the HIV Tat induces this effect. We demonstrate that increased IL-2 secretion is due to Tat-enhanced IL-2 promoter activation. Tat derepresses and activates the distal AP-1 site (position -185 to -177) in the IL-2 promoter. In nonstimulated T cells a repressor complex containing NF-IL6, JunB, c-Fos and Fra-1 is formed on the AP-1(IL-2/d) site and represses IL-2 promoter activity. After T cell activation, a heterodimeric activator containing p65 and c-Jun binds to the AP-1(IL-2/d) site. HIV Tat enhances activation of NF-kappaB and consequently, activates the AP-1(IL-2/d) site. Our data provide evidence for a novel mechanism by which HIV Tat dysregulates IL-2 production and therefore may contribute to the HIV-1 infection in a way yet to be clarified.

The role of Wilms' tumor suppressor 1 (WT1) in leukemogenesis has been investigated mostly in acute (AML) and chronic (CML) myeloid leukemias. So far, its oncogenic role has been controversially discussed because both overexpression and inactivating mutations are found. A recent study on primary samples from patients with acute T-cell leukemia (T-ALL) revealed that most of them do not express WT1 proteins although they express WT1 mRNA. In our study, we investigated WT-1 expression in ten T-ALL cell lines established from leukemia/lymphoma patients. We show that consistent with the finding in primary T-ALL cells, most of the leukemic T-cell lines tested do not overexpress WT1 proteins. We found that leukemic T-cells overexpressing WT1 protein produce higher levels of CD95L and show elevated CD95L-mediated activation-induced cell death (AICD) compared to cells lacking or expressing low levels of WT1. Ectopic expression of WT1 in the WT1-nonexpressing leukemic T-cell line increases CD95L expression and elevates activation-induced apoptosis, whereas silencing WT1 expression in the WT1-overexpressing leukemic T-cell line by siRNA confers reduced CD95L expression and reduction in AICD. Chromatin immunoprecipitation and luciferase-promoter reporter analysis demonstrate that WT1 binds to and enhances CD95L promoter activity through the Egr-binding sites. Our study provides a new role of WT1 in regulation of CD95L-mediated cell death.

The CD95 (also called APO-1 or Fas) system plays a major role in the induction of apoptosis in lymphoid and nonlymphoid tissues in response to a variety of extracellular signals, including chemotherapeutic drugs. Here we report that the CD95 ligand (CD95L) is upregulated in hepatoma cells upon treatment with antineoplastic drugs. Upregulation by different chemotherapeutic drugs is functionally relevant for drug-induced apoptosis and is mediated by transcriptional mechanisms. The MEKK1/JNKK pathway and a novel AP-1 element in the CD95L promoter downstream of the TATA box are required for CD95L upregulation. Thus, understanding the mechanisms of CD95-mediated apoptosis through CD95L upregulation upon treatment of hepatocellular carcinomas with chemotherapeutic drugs may contribute to the improvement of anticancer chemotherapy.

Efficient activation of antigen-specific T cells requires co-stimulatory signals provided e.g. by CD28. Re-exposure to antigen and CD28 co-stimulation reduces activation-induced cell death (AICD) and increases the number of T cells performing effector functions. AICD is mediated predominantly by CD95 (APO-1/Fas) and its cognate ligand (CD95L). In an in vitro model system, using human peripheral activated T cells, we demonstrate here that co-stimulation prevents CD95L expression. Moreover, we show that co-stimulation reduces the activity of the CD95 death-inducing signaling complex and procaspase-8 activation. In parallel, co-stimulation strongly increases expression of the short form of the FLICE-inhibitory protein c-FLIPshort and of Bcl-xL. These data provide important new insight into the molecular mechanisms of apoptosis resistance in co-stimulated T cells.

Background: Epidemiologic studies and experiments with mouse models suggest that polyaromatic hydrocarbons contained in, among others, diesel exhaust particles can promote the development of allergy. Objective: Because IL-4 organizes allergic responses in vivo, we have investigated whether pyrene, a major compound of diesel exhaust particles, can affect the production of IL-4. Methods: IL-4 production by primary human T cells was assessed by ELISA and messenger RNA transcription was detected by Northern blotting. Activation of the IL-4 promoter was tested in reporter gene assays with transiently transfected cell lines. Results: Pyrene induced transcription of IL-4 messenger RNA and expression of IL-4 protein in primary human T cells. Pyrene, but not related polyaromatic hydrocarbons, enhanced basal transcription of the human and mouse IL-4 promoter. Conclusion: Our results suggest that pyrene may promote allergic diseases by inducing the production of IL-4. (J Allergy Clin Immunol 2000;105:796-802.)

The secretion of IL-4, which displays many important immunoregulatory functions, is restricted to cells of the Th2 subtype. In this study, we investigated the early signaling events leading to the activation of IL-4 transcription. Vav, the protein kinase C (PKC) isoform Θ, and the adaptor protein SLP76 (SH2-domain-containing leukocyte protein of 76 kDa), induced transcription from the IL-4 promoter. Vav and PKCΘ synergistically activated human IL-4 promoter transcription and IL-4 mRNA production and were found to be constitutively associated in vivo. CD3/CD28-induced IL-4 transcription was inhibited upon coexpression of dominant negative forms of Vav, the adaptor proteins LAT (linker for activation of T cells) and SLP76, PKCΘ, and components of the pathways leading to the activation of c-Jun N-terminal kinase (mitogen-activated protein kinase kinase 7 (MKK7), mixed lineage kinase 3 (MLK3)) and NF-κB (IκB kinase α and IκB kinase β). The Vav/PKCΘ-mediated synergistic activation of IL-4 transcription was not inhibited by cyclosporin A. Three independent experimental approaches revealed that Vav/PKCΘ-derived signals selectively target the P1 and positive regulatory element (PRE)-I elements contained within the human IL-4 promoter. Vav/PKCΘ strongly activated a luciferase reporter construct controlled by trimerized P1 or PRE-I elements and furthermore stimulated DNA binding of nuclear proteins to the P1 and PRE-I elements. Vav/PKCΘ-induced transcription from the IL-4 promoter was almost completely abrogated by mutation of either the P1 or the PRE-I element within the entire IL-4 promoter.

CD95(APO-1 / Fas) ligand (CD95L) gene expression is critically involved in activation-induced T cell apoptosis. We and other have previously shown that HIV-1 Tat which is essential for efficient HIV gene expression sensitizes CD95-mediated apoptosis and up-regulates CD95L expression in T cells. In the present study we have investigated the regulatory mechanism for CD95L expression. Two NF-kappaB binding sites are localized at - 537 to - 521 and - 57 to - 47 (relative to the transcription start site) of the human CD95L promoter. We show that both elements bind to NF-kappaB and SP-1 transcription factors and NF-kappaB is involved in transactivation of the CD95L promoter upon T cell activation. Mutations at each NF-kappaB site by two base pair substitutions resulted in 30 - 70 % reduction of the promoter activity. The effect of Tat on the human CD95L promoter activity was mapped to the same sites. Mutation of each NF-kappaB site also impaired the effect of Tat on CD95L promotor activity. We also show that ectopic expression of Tat protein in Jurkat T cells greatly increases NF-kappaB binding to its target DNA. Our studies provide evidence that Tat-enhanced CD95L expression is regulated at least in part by the NF-kappaB sites of the promoter.

Expression of the CD95 (APO-1/Fas) ligand (CD95L) in activated T cells is a major cause of activation-induced T cell apoptosis. The transcription factors NF-AT and Egr-3 (a member of the immediate-early transcription factors involved in cellular growth and differentiation) have been implicated in activation of the CD95L promoter upon T cell activation. On the basis of DNase I footprinting, electrophoretic mobility shift assay, antibody supershift analysis and transfection studies, we have identified two novel Egr-binding sites 5' upstream of the previously identified Egr site. Mutation analysis of each Egr site shows that all three sites are important for full CD95L promoter activity. Strikingly, all Egr sites, including the previously identified Egr site, are adjacent to or overlap with DNA sequences homologous to NF-AT binding sites and confer T cell activation-induced, cyclosporin A-sensitive transcriptional activity. Antibody supershift analysis revealed that NF-AT and Egr proteins are the components of inducible DNA-binding complexes formed on the two novel Egr sites. Cotransfection experiments showed that Egr-1, Egr-3 and NF-AT display a cooperative and synergistic activation of transcription mediated by these three Egr/NF-AT composite regulatory elements. These findings provide further insight into the mechanisms involved in the regulation of the CD95L expression in response to T cell activation.

Chemotherapeutic drugs cause DNA damage and kill cancer cells mainly by apoptosis. p53 mediates apoptosis after DNA damage. To explore the pathway of p53-dependent cell death, we investigated if p53-dependent apoptosis after DNA damage is mediated by the CD95 (APO-1/Fas) receptor/ligand system. We investigated hepatoma, gastric cancer, colon cancer, and breast cancer cell lines upon treatment with different anticancer agents known to act via p53 accumulation. Cisplatin, mitomycin, methotrexate, mitoxantrone, doxorubicin, and bleomycin at concentrations present in the sera of patients during therapy led to an upregulation of both CD95 receptor and CD95 ligand. Induction of the CD95 ligand occurred in p53 wild-type (wt), p53 mutant (mt), and p53 deficient (p53−/−) cell lines and at wt and mt conformation of temperature-sensitive p53 mutants. In contrast, upregulation of the CD95 receptor was observed only in cells with wt p53, not in cells with mt or without any p53. Restitution of inducible wt p53 function restored the ability of p53−/− Hep3B cells to upregulate the CD95 receptor in response to anticancer drugs. This rendered the cells sensitive to CD95-mediated apoptosis. In an attempt to understand how CD95 expression is regulated by p53, we identified a p53-responsive element within the first intron of the CD95 gene, as well as three putative elements within the promoter. The intronic element conferred transcriptional activation by p53 and cooperated with p53-responsive elements in the promoter of the CD95 gene. wt p53 bound to and transactivated the CD95 gene, whereas mt p53 failed to induce apoptosis via activation of the CD95 gene. These observations provide a mechanistic explanation for the ability of p53 to contribute to tumor progression and to resistance of cancer cells to chemotherapy.

CD28 serves as a costimulatory cell surface molecule in T cell activation. CD28 signaling may also play a role in balancing the inflammatory/humoral (Th1/Th2) responses during an immune reaction. CD28 costimulation has been shown to promote the production of Th2 cytokines including interleukin (IL)-4, a key cytokine essential for Th2 differentiation and for the pathogenesis of allergic inflammation. In this study, we show that IL-4 mRNA and activity of the IL-4 promoter can be activated by the CD28 signal alone and are further augmented by CD28 costimulation of alpha-CD3- or mitogen-activated Jurkat T cells. Two important IL-4 enhancer elements, positive regulatory element (PRE)-I and P1, are found to respond to CD28 stimulation-induced transactivation. In contrast to the Th1 IL-2 CD28RE, activity of the IL-4 PRE-I and P1 can be induced by the CD28 signal alone. In correlation with CD28-induced transcriptional activation, AP-1 (c-Jun, JunD) and NF-kappaB/Rel (c-Rel, RelA) family members are found to bind to the two regulatory elements PRE-I and P1 upon CD28 stimulation. The data provide the first mapping of the CD28-responsive site in a Th2 cytokine gene, the IL-4 gene. They also show that the CD28 signal can directly activate a gene (e.g. IL-4) at the transcriptional level.

Interleukin-4 (IL-4) is a pleiotropic immunomodulatory cytokine secreted by T helper 2 cells. The IL-4 promoter contains multiple sites with DNA sequences homologous to the IL-2 NF-AT binding site. One of these sites—the P2 site—located between –173 and –150 was previously found to be flanked by two octamer-like motifs. NF-ATp/c and octamer proteins were suggested to bind to this region and to cooperatively activate the promoter activity ( Chuvpilo et al., 1993 ). To precisely analyze the P2-binding factors we used antibodies against NF-ATp, NF-ATc, Fos, Jun, Oct-1 and Oct-2 in EMSA. We show here that nuclear extracts from T-cells form two P2-binding complexes—a PMA/ionomycin-inducible and a constitutive one. The PMA/ionomycin-inducible complex contains NF-ATp/c, Fos and Jun. No octamer binding factors could be detected in either of the two complexes. Analysis of the precise DNA contact points of the two complexes showed that both complexes are formed in the center of the NF-AT consensus site. No DNA contact points could be detected in the octamer-like motif site. Furthermore, purified recombinant POU domains of Oct-1 and Oct-2 failed to bind to the P2 site, suggesting that this site is not an independent octamer-binding site. Therefore, the DNA sequence at –173 to –150 of the IL-4 promoter is a binding site for NF-ATp/c and AP-1. Octamer proteins are unlikely to cooperate with NF-ATp/c at this site. © 1997 Elsevier Science B.V. All rights reserved.

Targeting the cancer cell cycle machinery is an important strategy for cancer treatment. Cdc25A is an essential regulator of cycle progression and checkpoint response. Over-expression of Cdc25A occurs often in human cancers. In this study, we show that Rocaglamide-A (Roc-A), a natural anticancer compound isolated from the medicinal plant Aglaia, induces a rapid phosphorylation of Cdc25A and its subsequent degradation and, thereby, blocks cell cycle progression of tumor cells at the G1-S phase. Roc-A has previously been shown to inhibit tumor proliferation by blocking protein synthesis. In this study, we demonstrate that besides the translation inhibition Roc-A can induce a rapid degradation of Cdc25A by activation of the ATM/ATR-Chk1/Chk2 checkpoint pathway. However, Roc-A has no influence on cell cycle progression in proliferating normal T lymphocytes. Investigation of the molecular basis of tumor selectivity of Roc-A by a time-resolved microarray analysis of leukemic vs. proliferating normal T lymphocytes revealed that Roc-A activates different sets of genes in tumor cells compared with normal cells. In particular, Roc-A selectively stimulates a set of genes responsive to DNA replication stress in leukemic but not in normal T lymphocytes. These findings further support the development of Rocaglamide for antitumor therapy.

Hodgkin/Reed-Sternberg lymphoma (HL) is a clonal B-cell-related malignancy. Although many patients with HL can be cured by the current regimen of high-dose multi-agent chemotherapy, the treatment causes high risks of later pathologies including secondary malignancies. This fact highlights the demand to develop rational treatment for HL. Survival and growth of HL cells are largely dependent on their microenvironment. In this study, using the HL cell lines L1236 and KM-H2 as model systems, we investigated the role of IL-4/IL-13 signaling in regulation of drug sensitivity and resistance in HL. We show that specific blocking of IL-4 and IL-13-mediated STAT6 activation by either an IL-4-binding fusion protein APG598 or an IL-4R antagonist APG201 (R121D/Y124D) renders HL cells more prone to apoptotic killing by chemotherapeutic drugs such as Mitomycin C, 5-Fluorouracil, Etopside, Doxorubicin and Paclitaxel. This effect is due to inhibition of STAT6-mediated elevation of expression of the anti-apoptotic Bcl-2 family protein Bcl-xL. Employing ChIP analysis in combination with APG201 or STAT6-specific siRNA we identified a defined STAT6-binding site in the Bcl-xL promoter region from -1967 to -1957 of the transcription start site. Our data demonstrate that the IL-4/IL-13-STAT6-Bcl-xL axis may be an important target for HL treatment. This study also suggests that combination of classical chemotherapeutic drugs with the IL-4/IL-13 antagonists may enhance efficacy and reduce risks of toxicity from high dose of drugs in HL treatment.

Rocaglamide (1) is the parent cyclopenta[b]benzofuran derivative which was first identified as an antileukemic agent from the dried roots and stems of Aglaia elliptifolia Merr. (family Meliaceae) in 1982. Based on these findings, phytochemical interest in this genus increased sharply and up to date, more than 100 rocaglamide-type (= flavagline) compounds and structurally related derivatives have been identified from over 30 Aglaia species. Rocaglamide-type compounds show pronounced pharmacological activities including primarily (but not exclusively) antiproliferative and anti-inflammatory activity. The molecular mechanisms underlying these activities have been elucidated in recent years and established rocaglamide and several of its derivatives as interesting candidates for drug development especially in the field of anti-cancer research. Due to their unique structural features and promising pharmacological activities, several strategies leading to total synthesis of enantiomeric rocaglamide derivatives were developed and optimized. This chapter reviews the chemistry and biology of the rocaglamide-type derivatives and related compounds, with emphasis on their structural diversity, biosynthesis, pharmacological significance and total synthesis.