Triptolide, a diterpenoid isolated from the Chinese herb Tripterygium wilfordii Hook.f, has shown antitumor activities in a broad range of solid tumors. Here, we examined its effects on leukemic cells and found that, at 100 nM or less, it potently induced apoptosis in various leukemic cell lines and primary acute myeloid leukemia (AML) blasts. We then attempted to identify its mechanisms of action. Triptolide induced caspasedependent cell death accompanied by a significant decrease in XIAP levels. IntroductionTripterygium wilfordii Hook.f, a member of the Celastraceae family of plants, has been used in Chinese medicine for centuries. Triptolide, a diterpenoid, was first isolated from the plant and structurally characterized in 1972 1 and has been used for the treatment of a variety of autoimmune diseases and as an immunosuppressant in patients with organ and tissue transplantations. [1][2][3][4] Recently, triptolide was shown to have antitumor properties by suppressing the growth and inducing apoptosis of a broad range of human tumor cells. [5][6][7][8][9] Triptolide was also shown to sensitize cells to death induced by various agents, such as Apo2/Trail, TNF-␣, and different chemotherapeutic agents. [10][11][12] However, despite the recognized potent antitumor activity of triptolide, our knowledge regarding its mechanism of action is still limited. So far it is known only that triptolide blocks TNF-␣-mediated induction of c-IAP1 and c-IAP2, and the activation of NFB, 10,13,14 and induces caspase activation. 15,16 Acute myeloid leukemia (AML) is an aggressive hematologic malignancy. Despite major efforts during the past 30 years, limited progress has been made in the treatment of AML. The current primary treatment for AML is chemotherapy, which induces cell death mainly by apoptosis mediated by either the intrinsic mitochondrial or the extrinsic death receptor pathway, both of which lead to caspase activation and cell disintegration. The development of innovative therapies and identification of more effective drugs, therefore, remain high priorities for leukemia research. Because of its antitumor properties, triptolide holds promise as a treatment for leukemia. However, other than recent reports that triptolide induced apoptosis in U937 cells by activating caspase-3 and downregulating XIAP, 16 and that it down-regulated Bcr-Abl expression and induced apoptosis in K562 cells, 17 virtually no work has been done to elucidate the activity and mechanisms of triptolide in leukemia. Through an understanding of the molecular mechanisms that mediate the proapoptotic activity of triptolide, it will be possible to better understand its antileukemic effects and to determine whether it is a candidate for clinical use. In addition, knowing the molecular targets of triptolide and its mechanism of action will enable us to design rational combination therapies that more efficiently eradicate leukemic cells. In the study described here, we examined the effects of triptolide on various leukemic cell lines and primary AML blasts a...

UV-induced mutations in the p53 tumor suppressor gene play an essential role in skin cancer development. We report here that such mutations can be detected in UV-irradiated mouse skin months before the gross appearance of skin tumors. Application of SPF-15 sunscreens to mouse skin before each UV irradiation nearly abolished the frequency of p53 mutations. These results indicate that p53 mutation is an early event in UV skin carcinogenesis and that inhibition of this event may serve as an early end point for assessing protective measures against skin cancer development.

Biopsies of the alternate sites suggested by our simulation studies are feasible and reproducible. This new strategy significantly enhanced (p = 0.0075) prostate cancer detection compared to conventional sextant biopsies in men undergoing a repeat procedure.

Raf/MEK/Erk signaling is activated in the majority of acute myeloid leukemias (AMLs), providing rationale for targeting this pathway with therapeutic intent. We investigated growthinhibitory and proapoptotic effects of sorafenib in AML. Our studies demonstrated that sorafenib significantly inhibited the phosphorylation levels of Raf downstream target proteins MEK1/2 and Erk, induced apoptosis and inhibited colony formation in AML cell lines and in primary AML samples. Mechanistically, treatment with sorafenib resulted in upregulation of proapoptotic Bim, accompanied by an increase in Bad, Bax and Bak protein levels and decreased Mcl-1, X-linked inhibitor of apoptosis and surviving levels, which mainly led to the activation of the intrinsic apoptotic pathway. Silencing of Bim protein expression significantly abrogated sorafenibinduced apoptosis, suggesting a critical function of Bim in the activation of the intrinsic mitochondrial pathway induced by sorafenib. Importantly, sorafenib also modulated phospho-Erk, Bim, Bax and Mcl-1 levels in samples procured from patients in an ongoing Phase I clinical trial of sorafenib in AML. Combination of sorafenib with cytarabine or the novel small molecule Bcl-2 inhibitor ABT-737 synergistically induced cell death in AML cell lines. Our results strongly suggest potential activity of sorafenib as a novel mechanism-based therapeutic agent in AML.

Biopsies of the alternate sites suggested by our simulation studies are feasible and reproducible. This new strategy significantly enhanced (p = 0.0075) prostate cancer detection compared to conventional sextant biopsies in men undergoing a repeat procedure.

The equal distribution of chromosomes during mitosis is critical for maintaining the integrity of the genome. Essential to this process are the capture of spindle microtubules by kinetochores and the congression of chromosomes to the metaphase plate . Polo-like kinase 1 (Plk1) is a mitotic kinase that has been implicated in microtubule-kinetochore attachment, tension generation at kinetochores, tension-responsive signal transduction, and chromosome congression . The tension-sensitive substrates of Plk1 at the kinetochore are unknown. Here, we demonstrate that human Nuclear distribution protein C (NudC), a 42 kDa protein initially identified in Aspergillus nidulans and shown to be phosphorylated by Plk1 , plays a significant role in regulating kinetochore function. Plk1-phosphorylated NudC colocalizes with Plk1 at the outer plate of the kinetochore. Depletion of NudC reduced end-on microtubule attachments at kinetochores and resulted in defects in chromosome congression at the metaphase plate. Importantly, NudC-deficient cells exhibited mislocalization of Plk1 and the Kinesin-7 motor CENP-E from prometaphase kinetochores. Ectopic expression of wild-type NudC, but not NudC containing mutations in the Plk1 phosphorylation sites, recovered Plk1 localization at the kinetochore and rescued chromosome congression. Thus, NudC functions as both a substrate and a spatial regulator of Plk1 at the kinetochore to promote chromosome congression.

Surgical resection is the only curative strategy for pancreatic cancer (PC). Unfortunately, >80% of pancreatic cancer patients bear inoperable, locally advanced, chemoresistant tumors demonstrating the urgent need for development of novel therapeutic approaches to treat this disease. Here we report that the synthetic triterpenoid 2-cyano-3,12 dioxooleana-1,9 dien-28-imidazolide (CDDO-Im) antagonizes PC cell growth by inducing apoptosis at submicromolar concentrations. Notably, we demonstrate for the first time that the cytotoxicity of CDDO-Im is accompanied by the rapid and selective depletion of mitochondrial glutathione that results in accumulation of reactive oxygen species, oxidation of the cellular glutathione pool, loss of mitochondrial membrane potential, and phosphatidylserine externalization. The parent compound CDDO as well as the methyl ester of CDDO also depleted mitochondrial glutathione, demonstrating that this effect is mediated by the triterpenoid nucleus of these agents. Cotreatment with sulfhydryl nucleophiles completely prevented apoptosis and loss of viability induced by CDDO-Im, whereas alkylation of intracellular thiols by diethylmaleate or cotreatment with dithiothreitol decreased the accumulation of a biotinylated derivative of CDDO, TP-301, in PC cells, suggesting that intracellular reduced thiols are functional targets of the electrophilic triterpenoid nucleus of CDDO and its derivatives. In conclusion, our report is the first to identify mitochondrial glutathione as a target of CDDO and its derivatives and demonstrates that depletion of this antioxidant in the mitochondria is an effective strategy to induce cell death in PC cells. These results suggest that CDDO and its derivatives may offer a clinical benefit for the treatment of PC. Pancreatic cancer (PC)2 is a lethal disease characterized by a high metastatic potential and rapid progression with a median survival rate of only 24 weeks in untreated cases (1, 2). Tumor resection is still the most effective treatment for PC. However, because of local invasion and/or metastasis, only ϳ15-20% of PC patients qualify for surgical intervention. For locally advanced, unresectable, and metastatic disease, treatment is palliative at best and usually consists of 5-fluorouracil or gemcitabine alone or in combination with radiotherapy. Unfortunately, despite the moderate success of gemcitabine (2Ј,2Ј-difluorodeoxycitidine), median survival rates remain under 6 months for patients with metastatic disease (2-4). Given the poor performance of existing therapies, the development of novel therapies for the treatment of advanced metastatic pancreatic cancer is of utmost importance.Glutathione (GSH) is a critical intracellular antioxidant that scavenges reactive oxygen species (ROS) directly and indirectly by serving as cofactor for glutathione peroxidases, glutaredoxins, and lipid peroxidases (5, 6). Oxidized GSH (GSSG) must be converted back to GSH by the action of glutathione reductase, an enzyme that utilizes the pyridine nucleotide NADPH as a cofac...

Macrophage secretion of VEGF in response to the hypoxic tumor microenvironment contributes to tumor growth, angiogenesis, and metastasis. We have recently demonstrated that macrophages stimulated with GM-CSF at low O2 secrete high levels of a soluble form of the VEGF receptor (sVEGFR-1), which neutralizes VEGF and inhibits its biological activity. Using siRNA targeting to deplete HIF-1α or HIF-2α in murine macrophages, we found that macrophage production of sVEGFR-1 in response to low O2 was dependent on HIF-2α, while HIF-1α specifically regulated VEGF production. In our current report, we evaluated the growth of B16F10 malignant melanoma in mice with a monocyte/macrophage-selective deletion of HIF-1α or HIF-2α (HIF-1αflox/flox-or HIF-2αflox/+/LysMcre mice). GM-CSF treatment increased intra-tumoral VEGF and sVEGFR-1 in control mice, an effect that was associated with a decrease in microvessel density. GM-CSF treatment of HIF-1αflox/flox/LysMcre mice induced sVEGFR-1 but not VEGF, resulting in an overall greater reduction in tumor growth and angiogenesis compared to control mice. In addition, real-time PCR for melanoma-specific genes revealed a significantly reduced presence of lung micrometastases in HIF-1αflox/flox/LysMcre mice treated with GM-CSF. Conversely, GM-CSF treatment induced VEGF but not sVEGFR-1 in HIF-2αflox/+/LysMcre mice, and correspondingly, GM-CSF did not decrease tumor growth, angiogenesis, or lung metastasis in these mice. This study reveals opposing roles for the HIFs in the regulation of angiogenesis by tumor-associated macrophages, and suggests that administration of GM-CSF might be an effective means of inducing sVEGFR-1 and inhibiting tumor growth and angiogenesis in patients with melanoma.