Rebecca Köhler

The natural anticancer compounds rocaglamides inhibit the Raf-MEK-ERK pathway by targeting prohibitin 1 and 2.

Rocaglamides are potent natural anticancer products that inhibit proliferation of various cancer cells at nanomolar concentrations. We have recently shown that these compounds prevent tumor growth and sensitize resistant cancer cells to apoptosis by blocking the MEK-ERK-eIF4 pathway. However, their direct molecular target(s) remain(s) unknown. In this study, using an affinity chromatography approach we discovered that prohibitin (PHB) 1 and 2 are the direct targets of rocaglamides. Binding of rocaglamides to PHB prevents interaction between PHB and CRaf and, thereby, inhibits CRaf activation and subsequently CRaf-MEK-ERK signaling. Moreover, knockdown of PHB mimicked the effects of rocaglamides on the CRaf-MEK-ERK pathway and cell cycle progression. Thus, our finding suggests that rocaglamides are a new type of anticancer agent and that they may serve as a small-molecular tool for studying PHB-mediated cellular processes.

Wogonin and related natural flavones are inhibitors of CDK9 that induce apoptosis in cancer cells by transcriptional suppression of Mcl-1

The wogonin-containing herb Scutellaria baicalensis has successfully been used for curing various diseases in traditional Chinese medicine. Wogonin has been shown to induce apoptosis in different cancer cells and to suppress growth of human cancer xenografts in vivo. However, its direct targets remain unknown. In this study, we demonstrate for the first time that wogonin and structurally related natural flavones, for example, apigenin, chrysin and luteolin, are inhibitors of cyclin-dependent kinase 9 (CDK9) and block phosphorylation of the carboxy-terminal domain of RNA polymerase II at Ser2. This effect leads to reduced RNA synthesis and subsequently rapid downregulation of the short-lived anti-apoptotic protein myeloid cell leukemia 1 (Mcl-1) resulting in apoptosis induction in cancer cells. We show that genetic inhibition of Mcl-1 or CDK9 expression by siRNA is sufficient to mimic flavone-induced apoptosis. Pull-down and in silico docking studies demonstrate that wogonin directly binds to CDK9, presumably to the ATP-binding pocket. In contrast, wogonin does not inhibit CDK2, CDK4 and CDK6 at doses that inhibit CDK9 activity. Furthermore, we show that wogonin preferentially inhibits CDK9 in malignant compared with normal lymphocytes. Thus, our study reveals a new mechanism of anti-cancer action of natural flavones and supports CDK9 as a therapeutic target in oncology.

Wogonin and Related Natural Flavones Overcome Tumor Necrosis Factor-related Apoptosis-inducing Ligand (TRAIL) Protein Resistance of Tumors by Down-regulation of c-FLIP Protein and Up-regulation of TRAIL Receptor 2 Expression

Background: Most primary cancer cells are resistant to TRAIL-mediated apoptosis. Results: Wogonin and related natural flavones suppress c-FLIP but up-regulate TRAIL receptor-2 expression and thereby sensitize resistant tumor cells to TRAIL-mediated apoptosis. Conclusion: Wogonin and related natural flavones can overcome TRAIL resistance of cancer cells. Significance: These flavones can be developed as adjuvants for TRAIL-based anticancer therapy. 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.

Rocaglamide breaks TRAIL resistance in HTLV-1-associated adult T-cell leukemia/lymphoma by translational suppression of c-FLIP expression.

The human T-cell leukemia virus type-1 (HTLV-1)-associated adult T-cell leukemia/lymphoma (ATL) is incurable by currently known therapies. ATL samples and cell lines derived from ATL patients show restricted sensitivity to tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) and CD95 ligand (CD95L). We have recently shown that HTLV-1-infected cells express elevated levels of cellular caspase-8 FLICE-inhibitory protein (c-FLIP) conferring resistance to receptor-mediated apoptosis. This finding underscores the demand to develop new strategies for treatment of ATL. In this study, we show that the naturally occurring herbal compound Rocaglamide (Roc) sensitizes CD95L- and TRAIL-induced apoptosis in HTLV-1-infected cells by downregulation of c-FLIP expression. Investigation of the molecular mechanism of Roc-mediated downregulation of c-FLIP revealed that it inhibits phosphorylation of the translation initiation factor 4E (eIF4E), a key factor that controls the rate-limiting step of translation, through inhibition of the MEK–ERK–MNK1 signaling pathway. This event prevents de novo synthesis of short-lived proteins such as c-FLIP in HTLV-1-infected cells. Our data suggest that Roc may serve as an adjuvant for TRAIL-based anticancer therapy.

Early Growth Response Protein-1 (Egr-1) Is Preferentially Expressed in T Helper Type 2 (Th2) Cells and Is Involved in Acute Transcription of the Th2 Cytokine Interleukin-4

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-κB cooperatively bind to an NFAT/NF-κB-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.

Rocaglamide sensitizes leukemic T cells to activation-induced cell death by differential regulation of CD95L and c-FLIP expression

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.

Curcumin Suppresses T Cell Activation by Blocking Ca2+ Mobilization and Nuclear Factor of Activated T Cells (NFAT) Activation

Background: Curcumin can overcome CsA resistance. However, the molecular mechanism is unknown. Results: Curcumin blocks T cell stimulation-induced Ca2+ mobilization and thereby prevents NFAT activation, a mechanism different from CsA. Conclusion: Curcumin is an immunosuppressive phytochemical that blocks Ca2+ signaling. Significance: The study demonstrates for the first time that curcumin is a potent inhibitor of NFAT activation via blocking Ca2+ signaling in T cells. 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 Ca2+ mobilization with IC50 = ∼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 Ca2+ 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.

Targeting CDK9 by wogonin and related natural flavones potentiates the anti-cancer efficacy of the Bcl-2 family inhibitor ABT-263

Tumor initiation, progression and resistance to therapies are tightly associated with over‐expression of anti‐apoptotic proteins Bcl‐2, Bcl‐xL, 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‐xL, 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‐xL 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.

Targeting the IL‐4/IL‐13 signaling pathway sensitizes Hodgkin lymphoma cells to chemotherapeutic drugs

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 and a XIAP inhibitor cooperatively sensitize TRAIL-mediated apoptosis in Hodgkin's lymphomas

Although most of the patients with Hodgkins lymphoma (HL) can be cured by the current regimen of high‐dose multiagent chemotherapy, the treatment causes high risks of later toxicities including secondary malignancies. Therefore, new rational strategies are needed for HL treatment. Tumor necrosis factor‐related apoptosis‐inducing ligand (TRAIL) is a promising anticancer agent due to its tumor selectivity and its lack of toxicity for normal cells. Unfortunately, many cancers remain resistant to TRAIL including HL. HL is characterized by enhanced expression of cellular caspase‐8 (FLICE)‐inhibitory protein (c‐FLIP) and X‐linked inhibitor of apoptosis (XIAP), which block receptor‐mediated apoptosis by inhibiting caspase‐8 and caspase‐3, respectively. We have recently discovered the herbal compound Rocaglamide, which breaks TRAIL‐resistance in acute T cell leukemia through inhibition of c‐FLIP expression. We have also shown that small molecule XIAP inhibitors can sensitize TRAIL‐mediated apoptosis in several resistant tumors. However, whether targeting XIAP or c‐FLIP is also a suitable strategy to prime HL cells for TRAIL‐induced apoptosis has not yet been investigated. In our study, we show that Rocaglamide suppresses c‐FLIP expression in HL cells in a dose‐ and time‐dependent manner. However, downregulation of c‐FLIP alone was not sufficient to sensitize TRAIL‐induced apoptosis in HL cells. Similarly, treatment of HL cells with a small molecule XIAP inhibitor resulted in a moderate induction of apoptosis. However, inhibition of XIAP alone was also not sufficient to enhance TRAIL‐induced cell death. Synergistic increase in TRAIL‐mediated killing of HL cells was only obtained by combination of Rocaglamide and XIAP inhibitors. Our study demonstrates that targeting both c‐FLIP and XIAP are necessary for an efficient treatment of HL.