Introduction

Failure in induction of apoptosis often causes resistance of tumors to chemotherapy. To develop rational strategies for cancer treatment, we focus on exploring and understanding the molecular mechanisms by which tumors escape from induction of apoptosis. HTLV-1-associated adult T-cell leukemia (ATL) is incurable by currently known therapies largely due to apoptosis resistance. We have shown that the HTLV-1 transactivator Tax is responsible for the resistance to death receptor-mediated apoptosis by elevation of c-FLIP expression (1). Hodgkin lymphoma (HL) is another unique malignancy whose growth relies on cytokines in the microenvironment. Although most patients with HL can be cured by the current regimen of high-dose multi-agent chemotherapy, the treatment causes a high risk of later toxicities including secondary malignancies. With an increasing cancer rate worldwide there is an urgent need for the improvement of anti-cancer drugs. Inspection of the developmental process of drugs during the past half century demonstrates that natural products are an important source of drugs in medicine. In fact, about two thirds of anti-cancer drugs approved today are either natural products or developed on the basis of knowledge gained from natural products. Therefore, our recent studies are also focusing on discovering new anti-cancer drugs from the traditional Chinese medicine (TCM).

Discovery of new anti-cancer drugs from natural sources

Apoptosis induction by defined compounds derived from Traditional Chinese Medicine (TCM): TCM compounds selectively target cancer cells via suppression of the anti-apoptotic protein Mcl-1, differential regulation of the redox status and activities of the stress MAPK p38, JNK and the survival MAPK ERK, activation of PLCγ; and Ca2+ signals. These events lead to induction of mitochondria-mediated cell death. TCM compounds may also sensitize receptor-mediated apoptosis in tumor cells by inhibition of c-FLIP and XIAP and up-regulation of CD95L and TRAIL receptor expression.
© Dr. Min Li-Weber

Recently, TCM has gained considerable attention as a new source of anti-cancer drugs. We have identified two natural anti-cancer compounds, Wogonin and Rocaglamide, from herbs used in TCM, which preferentially induce apoptosis in tumor cells in vitro and in vivo. Importantly, both compounds show no or very little toxicity to normal cells (2-4, 7, 8). Investigation of the selective anti-tumor effects of Wogonin revealed two mechanisms: (I) Tumor cells show an increased metabolism, often produce elevated levels of ROS and display an altered redox status. Wogonin is a strong anti-oxidant that differentially regulates the redox status of tumor and normal cells by neutralizing .O2- to the more reduced form H2O2. This effect leads to stronger activation of PLCγ in malignant T cells and, consequently, triggers prolonged (and/or stronger) intracellular Ca2+ mobilization and Ca2+-mediated apoptosis (3). (II) The cyclin-dependent kinase 9 (CDK9), which has recently been suggested as a promising therapeutic target in oncology and virology, is a direct molecular target of Wogonin and related natural flavones (8). Inhibition of CDK9 activity by these flavones results in reduced RNA synthesis and subsequently rapid down-regulation of the short-lived anti-apoptotic protein Mcl-1, leading to apoptosis induction in cancer cells (8). We also show that Rocaglamide preferentially kill tumor cells by differential activation of the MAPK pathways in malignant vs. normal T cells (2).

Exploration of rational therapeutic strategies for cancer treatment

The therapeutic use of some drugs is limited due to their toxic side effects and/or low efficiency. Therefore, new rational strategies are needed for cancer treatment. For example, although TRAIL is a promising anticancer agent due to its tumor selectivity and its lack of toxicity to normal cells, many tumors are TRAIL-resistant. HTLV-1-associated ATL is resistant to receptor-mediated apoptosis due to over-expression of c-FLIP (1). To overcome this obstacle, we treated these cells with Rocaglamide or Wogonin to block c-FLIP expression at the translational or the transcriptional level, respectively. We demonstrated that both compounds overcome TRAIL resistance in ATL cells (7). Another example is HL. HL is characterized by enhanced expression of c-FLIP and XIAP, which blocks receptor-mediated apoptosis by inhibiting caspase-8 and caspase-3, respectively. We show that Rocaglamide suppresses c-FLIP expression in HL cells in a dose- and time-dependent manner. However, down-regulation of c-FLIP alone was not sufficient to increase 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 a combination of Rocaglamide and XIAP inhibitors (9). Our study demonstrates that targeting both c-FLIP and XIAP is necessary for an efficient treatment of HL. In addition, we confirmed that HL cells express the high affinity IL-4 receptor and IL-4 promotes the survival of HL cells.  Inhibition of the IL-4 signaling pathway by an IL-4R (both type I and II) specific blocker results in sensitization of HL cells to apoptosis.

Regulation of IL-4 gene expression

Since the particular type of cytokines produced during an immune response determines the recruitment and activation of other immune cells, the differential production of cytokines is a critical determinant for the character of the immune response. IL-4 plays an important role in directing Th2 cell development and preventing the differentiation of Th1 cells. An unbalanced Th1/Th2 ratio is seen in many immunological diseases, including infectious diseases, autoimmune and allergic responses. During the last 20 years we have investigated the transcriptional control of IL-4 gene expression and have identified important transcription factors for the inducible activation of the IL-4 gene. We have recently identified two additional transcription factors, the lymphoid enhancer-binding factor 1 (LEF-1) and the early growth response gene product 1 (Egr-1), which are differentially expressed in Th1 and Th2 cells and are involved in negative and positive regulation of IL-4 gene expression, respectively (5, 6).

Key References

  1. Krueger A, Fas SC, Giaisi M, Bleumink M, Merling A, Stumpf C, Baumann S, Holtkotte D, Bosch V, Krammer PH and Li-Weber M. (2006) HTLV-1 tax protects against CD95-mediated apoptosis by induction of the cellular FLICE-inhibitory protein (c-FLIP). Blood 2006; 107:3933-3939. 
  2. Zhu JY, Lavrik  IN, Mahlknecht U, Giaisi M, Proksch P, Krammer PH and Li-Weber M. (2007) The traditional Chinese herbal compound Rocaglamide preferentially induces apoptosis in leukemia cells by modulation of MAPK activities. Int J Cancer 121, 1839-1846.
  3. Baumann S, Fas SC, Giaisi M, Müller WW, Merling A, Gülow K,  Edler L, Krammer PH and Li-Weber M. (2008) Wogonin preferentially kills malignant lymphocytes and suppresses T-cell tumor growth by inducing PLCg1- and Ca2+-dependent apoptosis. Blood 111, 2354-2363.       
  4. Zhu JY, Giaisi M, Köhler R, Wolfgang WW, Mühleisen A, Proksch P, Krammer PH and Li-Weber M. (2009) Rocaglamide sensitizes leukemic T cells to activation-induced cell death by differential regulation of CD95L and c-FLIP expression. Cell Death Differ 16, 1289-1299.
  5. Hebenstreit D, Giaisi M, Treiber MK, Zhang X, Mi H, Horejs-Hoeck J Krammer PH, Duschl A and Li-Weber M (2008). LEF-1 negatively controls interleukin-4 expression through a proximal promoter regulatory element. J Biol Chem 283, 22490-22497.
  6. Lohof M., Giaisi M, Köhler R, Casper B, Krammer PH and Li-Weber M. (2010) The early growth response protein-1 (Egr-1) is preferentially expressed in Th2 cell and is involved in acute transcription of the Th2 cytokine interleukin-4. J Biol Chem 285: 1643-1652.
  7. Bleumink M, Köhler R, Giaisi M, Proksch P, Krammer PH and Li-Weber M. (2011) Rocaglamide breaks TRAIL resistance in HTLV-1-associated adult T-cell leukemia/lymphoma by translational suppression of c-FLIP expression. Cell Death Differ 18, 362-370.
  8. Polier, G., Ding, J., Konkimalla, BV., Eick, D., Ribeiro N., Köhler, R., Giaisi, M., Efferth, T., Desaubry, L., Krammer, PH., Li-Weber, M. (2011) Wogonin and related natural flavones are inhibitors of CDK9 that induce apoptosis in cancer cells by transcriptional suppression of Mcl-1. Cell Death Disease, Jul 21;2:e182. doi: 10.1038
  9. Giaisi, M., Köhler, R., Fulda, S., Krammer, P.H. and Li-Weber, M. (2011) Rocaglamide and a XIAP inhibitor cooperatively sensitize TRAIL-mediated apoptosis in Hodgkin lymphomas. Int J Cancer, Sep 27. doi: 10.1002/ijc.26458. [Epub ahead of print].  

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