The traditional Chinese medical compound Rocaglamide protects nonmalignant primary cells from DNA damage-induced toxicity by inhibition of p53 expression.

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.

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 Ser(2). 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.

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.

The anti-inflammatory sesquiterpene lactone parthenolide suppresses CD95-mediated activation-induced-cell-death in T-cells.

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.

Fractionated gamma-irradiation renders tumour cells more responsive to apoptotic signals through CD95.

Signals through the CD95 surface receptor can specifically induce apoptosis. Some tumour cell lines are sensitive to CD95 signals, and insensitive cells can be converted to a sensitive phenotype if given appropriate treatment. To determine whether the apoptotic response of tumour cells to signalling through CD95 might be enhanced by ionizing irradiation, carcinoma cells were treated with either single-dose or fractionated gamma-irradiation. The response to treatment with an agonist anti-CD95 antibody was enhanced by pretreatment with either a single large dose or daily fractionated radiation. Fractionated irradiation induced cumulative and prolonged up-regulation of CD95 expression in cell lines bearing functional p53. Since two of four cell lines exhibiting heightened responsiveness to CD95-mediated signals following fractionated irradiation express mutant p53 and displayed little or no up-regulation of CD95, enhanced responsiveness did not correlate with p53 status and CD95 up-regulation. Continuous inhibition of CD95/CD95-ligand interactions during fractionated irradiation provided no protective effect to cells, arguing that autologous CD95/CD95-ligand interactions did not contribute to the direct lethal effect of irradiation. We conclude that fractionated gamma-irradiation provides an extended period of time when carcinoma cells are more responsive to CD95-mediated signals in vitro.

Surface expression of TRAIL/Apo-2 ligand in activated mouse T and B cells.

Like other members of the TNF family, TRAIL/Apo-2 ligand induces apoptosis in sensitive target cells in a caspase-dependent fashion. We recently found that TRAIL may be constitutively expressed on the surface of mouse and human tumor cells of T and B origin. To define the pattern of TRAIL expression in normal immune cells, freshly isolated splenocytes, Concanavalin A/IL-2-activated T cells and lipopolysaccharide-activated B cells were analyzed by surface staining with or without secondary stimulation. Activated, but not resting, CD3+ cells expressed TRAIL in an activation-dependent fashion. Conversely, freshly isolated B220+ cells displayed surface TRAIL and CD95L that were retained following activation. Restimulation with the protein kinase C activator phorbol 12-myristate 13-acetate and the calcium ionophore ionomycin or an agonistic anti-CD3 monoclonal antibody induced significant up-regulation of surface TRAIL and CD95L in CD3+, TCRalphabeta cells with CD4+ or CD8+ phenotype. Similarly to CD95L, TRAIL up-regulation was protein synthesis dependent and cyclosporin A sensitive. These results indicate that both TRAIL and CD95L are displayed on the cell surface of activated immune cells and may thus represent complementary effector pathways in the regulatory functions of T and B cells.

S-adenosylhomocysteine as a physiological modulator of Apo-1-mediated apoptosis.

APO-1/Fas (CD95) is a member of the tumor necrosis factor/nerve growth factor receptor superfamily and mediates apoptosis in various cell types. Here we show that L929 cells, expressing human APO-1 treated with agonistic antibodies (anti-APO-1), elicit an early and transient increase of S-adenosylhomocysteine (AdoHcy), a potent inhibitor of S-adenosylmethionine (AdoMet)-dependent methylation reactions. In contrast, anti-APO-1 did not induce an AdoHcy increase in L929-APO-1 Delta4 cells expressing a C-terminally truncated APO-1 lacking part of the 'death domain' known to be required for the transduction of apoptotic signals. Addition of adenosine and D, L-homocysteine also led to an increase of cellular AdoHcy thus enhancing anti-APO-1-induced killing of L929-APO-1 cells. Treatment with anti-APO-1 also induced release of arachidonic acid from phospholipids: this effect was augmented by elevated levels of AdoHcy. In contrast, AdoHcy had only a minor effect on anti-APO-1-mediated DNA fragmentation. These findings suggest that AdoHcy functions as a physiological modulator of APO-1-mediated cell death in L929 cells and enhances anti-APO-1-induced cell killing at least partially by acting via the phospholipase A2 pathway.