As part of the screening program for anticancer agents from natural sources, the sesquiterpene lactone goyazensolide (GZL) was identified as a potent NF-κB inhibitor. The hollow-fiber assay was used to evaluate the anti-tumor efficacy of GZL in vivo. The mechanistic effects of GZL were evaluated in the HT-29 colonic cell line to reveal the pathway through which GZL exerts its effects. NF-κB subunits p65 and p50 were inhibited, and the upstream mediator IκB kinase (IKKβ) was downregulated in a dose-dependent manner. Apoptosis was mediated by caspase-3, and cell cycle arrest was detected in G1-phase. Consequently, 96% of the cell population was in sub G1-phase after treatment with GZL (10 μM).The antitumor effect of GZL was observed at a dose of 12.5 mg/kg. Cell adhesion was affected as a result of NF-κB inhibition. GZL appears to selectively target the transcription factor NF-κB. In summary, GZL sensitizes HT-29 colon cancer cells to apoptosis and cell death in a dose-dependent manner both in vivo and in vitro, through NF-κB inhibition (IC50 = 3.8 μM). Thus, it is a new potent lead compound for further development into a new effective chemotherapeutic agent.

Isocitrate dehydrogenase 1 (IDH1), a cytosolic enzyme that converts isocitrate to alpha‐ketoglutarate, has been shown to be dysregulated during tumorigenesis. However, at what stage of cancer development IDH1 is dysregulated and how IDH1 may affect cell transformation and tumor promotion during early stages of cancer development are unclear. We used a skin cell transformation model and mouse skin epidermal tissues to study the role of IDH1 in early skin tumorigenesis. Our studies demonstrate that both the tumor promoter TPA and UVC irradiation decreased expression and activity levels of IDH1, not IDH2, in the tumor promotable JB6 P+ cell model. Skin epidermal tissues treated with dimethylbenz[α]anthracene/TPA also showed decreases in IDH1 expression and activity. In non‐promotable JB6 P‐cells, IDH1 was upregulated upon TPA treatment, whereas IDH2 was maintained at similar levels with TPA treatment. Interestingly, IDH1 knockdown enhanced, whereas IDH1 overexpression suppressed, TPA‐induced cell transformation. Finally, manganese superoxide dismutase overexpression suppressed tumor promoter induced decreases in IDH1 expression and mitochondrial respiration, while intracellular alpha‐ketoglutarate levels were unchanged. These results suggest that decreased IDH1 expression in early stage skin tumorigenesis is highly correlated with tumor promotion. In addition, oxidative stress might contribute to IDH1 inactivation, because manganese superoxide dismutase, a mitochondrial antioxidant enzyme, blocked decreases in IDH1 expression and activity. (Cancer Sci, doi: 10.1111/j.1349‐7006.2012.02317.x, 2012)

The compound 13-acetoxyrolandrolide was previously isolated from Rolandra fruticosa (L.) Kuntze (Asteraceae). Potent cytotoxic activity was found against HT-29 colon cancer cells compared with paclitaxel, EC50=0.16µM and EC50=0.0006µM, respectively. Initial screening showed that the NF-κB inhibition was IC50=7.1µM. The mechanism of action through which transcription factor NF-κB was further investigated and the expression of up-stream mediators such as IKKα and IKKβ was analyzed by immunoblotting. In this study, 13-acetoxyrolandrolide demonstrated similar effects to staurosporine, inducing loss of the mitochondrial membrane potential (ΨΔm). Cell cycle analysis showed a significant increase of HT-29 cells in the G1-phase after treatment, and 68% of treated cells were found in this G1-phase compared to 55% in the untreated cells. In addition, high intracellular levels of ROS were also detected in treated cells. These findings suggest that the mitochondrial activity of cancer cells was affected and NF-κB was inhibited, possibly through an oxidative pathway. Thus, chemical optimization of 13-acetoxyrolandrolide might lead to the discovery of a new potential cancer chemotherapeutic agent for the treatment of colon cancer.

Differentiated cells primarily metabolize glucose for energy via the tricarboxylic acid cycle and oxidative phosphorylation, but cancer cells thrive on a different mechanism to produce energy, characterized as the Warburg effect, which describes the increased dependence on aerobic glycolysis. The M2 isoform of pyruvate kinase (PKM2), which is responsible for catalyzing the final step of aerobic glycolysis, is highly expressed in cancer cells and may contribute to the Warburg effect. However, whether PKM2 plays a contributing role during early cancer development is unclear. In our studies, we have made an attempt to elucidate the effects of varying mitochondrial respiration substrates on skin cell transformation and expression of PKM2. Tumorigenicity in murine skin epidermal JB6 P+ (promotable) cells was measured in a soft agar assay using 12-O-tetradecanoylphorbol-13-acetate (TPA) as a tumor promoter. We observed a significant reduction in cell transformation upon pretreatment with the mitochondrial respiration substrate succinate or malate/pyruvate. We observed that increased expression and activity of PKM2 in TPA-treated JB6 P+ cells and pretreatment with succinate or malate/pyruvate suppressed the effects. In addition, TPA treatment also induced PKM2 whereas PKM1 expression was suppressed in mouse skin epidermal tissues in vivo. In comparison with JB6 P+ cells, the nonpromotable JB6 P− cells showed no increase in PKM2 expression or activity upon TPA treatment. Knockdown of PKM2 using a siRNA approach significantly reduced skin cell transformation. Thus, our results suggest that PKM2 activation could be an early event and play a contributing role in skin tumorigenesis. Cancer Prev Res; 4(9); 1476–84. ©2011 AACR.

Proceedings: AACR 102nd Annual Meeting 2011‐‐ Apr 2‐6, 2011; Orlando, FL Goyazensolide (C19H20O7) is a rare sesquiterpene lactone isolated from Piptocoma rufescens Cass. (Asteraceae). Goyazensolide significantly inhibits NF-κB transcription factor and decreased the cell growth of HT-29 colon cancer cells in a dose-dependent manner. In the present study, we investigated the effects of goyazensolide in a hollow fiber assay using in vivo setting. We also examined the involvement of ROS-signaling components (ROS levels, NF-κB transcription factor, mitochondrial membrane potential) and demonstrated that the antiproliferative effect of cancer cells is partially mediated by induction of oxidative stress. It is our aim to further investigate the molecular mechanism through which goyazensolide exerts its cytotoxic effect in vitro and its antitumor effect in vivo. Our data therefore supports goyazensolide as an active anti-proliferative agent with great potential for further development. Acknowledgment This research was supported by Program Project Grant P01-CA125066 funded by the National Cancer Institute, NIH.We also wish to acknowledge the plant taxonomists who collected the plant material used in this investigation. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 4040. doi:10.1158/1538-7445.AM2011-4040

Cancer cells distinguish themselves from normal cells in multiple ways. One such distinction is that cancer cells predominantly produce energy by a high rate of glycolysis followed by lactic acid fermentation even in the presence of oxygen, known as the “Warburg Effect”. Although glycolysis is inefficient to produce ATP compared with oxidative phosphorylation, metabolic intermediates produced during glycolysis may provide building blocks for cancer cells. Associated with this metabolic switch is up- or down-regulation of important metabolic enzymes, such as the M1 and M2 isoforms of pyruvate kinase (PKM1; PKM2) and isocitrate dehydrogenase 1 and 2 (IDH1; IDH2). PKM2 is highly expressed and critical for proliferation of tumor cells. Our initial studies showed that both UV irradiation and tumor promoters altered cellular metabolism via upregulating the activity and expression levels of PKM2 and downregulating PKM1. However, this M1/M2 shift has not been reported in early stage of cancer development. IDH1, a cytosolic enzyme which converts isocitrate to alpha-ketoglutarate, was recently discovered to be mutated in human brain cancer and leukemia. Interestingly, wild-type IDH1 produces alpha-ketoglutarate; however, mutant IDH1 generates 2-hydroxyglutarate, a known “oncometabolite.” We have found that decreased expression and activity levels of IDH1, not IDH2 were seen both in vitro in the tumor promotable JB6 P+ cell model and in vivo using mouse skin epidermal tissues, following UV and 12–0-tetradecanoylphorbol 13-acetate (TPA) treatment. More importantly, knockdown of IDH1 enhanced whereas overexpression of IDH1 suppressed cell transformation induced by tumor promoter TPA. During tumorigenesis, oncogenic activation can also generate oxidative stress. Since metabolism is the major source for ROS (reactive oxygen species) production, metabolic changes often observed in cancer cells may also be regulated by oxidative stress. Our initial studies revealed that PKM2 was upregulated and IDH1 downregulated in promotable mouse skin epidermal JB6 P+ cells, not in non-promotable JB6 P-cells. We have observed in previous studies that MnSOD expression/activity is lower in P+ cells, compared to P-cells, thus providing evidence that PKM2 activation and IDH1 inactivation may be regulated by oxidative stress. Lastly, overexpression of manganese superoxide dismutase (MnSOD) suppressed tumor promoter-induced PKM2 activation and decreased IDH1 expression. These results suggest that oxidative stress may contribute to PKM2 activation and IDH1 inactivation during early stage tumor promotion, suggesting mediation with MnSOD, a mitochondrial antioxidant enzyme, may play a cancer preventative role in regulating cellular metabolism. Citation Information: Cancer Prev Res 2011;4(10 Suppl):B59.