No abstract

As chronic myeloid leukemia (CML) progresses from the chronic phase to blast crisis, the levels of BCR-ABL increase. In addition, blast transformed leukemic cells display enhanced resistance to imatinib in the absence of BCR-ABL resistance mutations. Here we show that when BCR-ABL transformed cell lines were selected for imatinib resistance in vitro, the cells that grew out displayed higher BCR-ABL expression comparable to increase seen in accelerated forms of the disease. This enhanced expression of BCR-ABL was associated with an increased rate of glycolysis but a decreased rate of proliferation. The higher level of BCR-ABL expression in the selected cells correlated with a non-hypoxic induction of HIF-1α that was required for cells to tolerate enhanced BCR-ABL signaling. HIF-1α induction resulted in an enhanced rate of glycolysis but reduced glucose flux through both the TCA cycle and the oxidative arm of the pentose phosphate pathway (PPP). The reduction in oxidative PPP mediated ribose synthesis was compensated by the HIF-1α-dependent activation of the non-oxidative PPP enzyme, transketolase, in imatinib-resistant CML cells. In both primary cultures of cells from patients exhibiting blast transformation and in vivo xenograft tumors, use of oxythiamine which can inhibit both the pyruvate dehydrogenase complex and transketolase resulted in enhanced imatinib sensitivity of tumor cells. Together, these results suggest that oxythiamine can enhance imatinib efficacy in patients that present in the accelerated form of the disease.

Cervical cancer is the second most common malignancy among women worldwide and is highly radioresistant, often resulting in local treatment failure. For locally advanced disease, radiation is combined with low-dose chemotherapy; however, this modality often leads to severe toxicity. Curcumin, a polyphenol extracted from rhizomes of the plant Curcuma longa, is a widely studied chemopreventive agent that was shown to have a low toxicity profile in three human clinical trials. Here, we show that pretreatment of two cervical carcinoma cell lines, HeLa and SiHa, with curcumin before ionizing radiation (IR) resulted in significant dosedependent radiosensitization of these cells. It is noteworthy that curcumin failed to radiosensitize normal human diploid fibroblasts. Although in tumor cells, curcumin did not significantly affect IR-induced activation of AKT and nuclear factor-B, we found that it caused a significant increase in the production of reactive oxygen species, which further led to sustained extracellular signal-regulated kinase (ERK) 1/2 activation. The antioxidant compound N-acetylcysteine blocked the curcumin-induced increased reactive oxygen species (ROS), sustained activation of ERK1/2, and decreased survival after IR in HeLa cells, implicating a ROS-dependent mechanism for curcumin radiosensitivity. Moreover, PD98059 (2Ј-amino-3Ј-methoxyflavone)-, PD184352-[2-(2-chloro-4-iodo-phenylamino)-Ncyclopropylmethoxy-3,4-difluoro-benzamide], and U0126 [1,4-diamino-2,3-dicyano-1,4-bis(2-aminophynylthio)butadiene]-specific inhibitors of mitogen-activated protein kinase kinase 1/2 (MEK1/2) blocked curcumin-mediated radiosensitization, demonstrating that the sustained ERK1/2 activation resulting from ROS generation leads to curcumin-mediated radiosensitization. Together, these results suggest a novel mechanism for curcuminmediated radiosensitization involving increased ROS and ERK1/2 activation and suggest that curcumin application (either systemically or topically) may be an effective radiation modifying modality in the treatment of cervical cancer.

Tamoxifen is the most widely used adjuvant chemotherapeutic for the treatment of estrogen receptor (ER) positive breast cancer, yet a large body of clinical and preclinical data indicates that tamoxifen can modulate multiple cellular processes independently of ER status. Here, we describe the ER-independent effects of tamoxifen on tumor metabolism. Using combined pharmacological and genetic knockout approaches, we demonstrate that tamoxifen inhibits oxygen consumption via inhibition of mitochondrial complex I, resulting in an increase in the AMP/ATP ratio and activation of the AMPK signaling pathway in vitro and in vivo. AMPK in turn promotes glycolysis, and alters fatty acid metabolism. We also show that tamoxifen-induced cytotoxicity is modulated by isoform-specific effects of AMPK signaling, in which AMPKα1 promotes cell death through inhibition of the mTOR pathway and translation. By using agents which concurrently target distinct adaptive responses to tamoxifen-mediated metabolic reprogramming, we demonstrate increased cytotoxicity through synergistic therapeutic approaches. Our results demonstrate novel metabolic perturbations by tamoxifen in tumor cells which can be exploited to expand the therapeutic potential of tamoxifen treatment beyond ER+ breast cancer.

The initial and rate-limiting enzyme of the oxidative pentose phosphate shunt, glucose-6-phosphate dehydrogenase (G6PD), is inhibited by NADPH and stimulated by NADP(+). Hence, under normal growth conditions, where NADPH levels exceed NADP(+) levels by as much as 100-fold, the activity of the pentose phosphate cycle is extremely low. However, during oxidant stress, pentose phosphate cycle activity can increase by as much as 200-fold over basal levels, to maintain the cytosolic reducing environment. G6PD-deficient (G6PD(-)) cell lines are sensitive to toxicity induced by chemical oxidants and ionizing radiation. Compared to wild-type CHO cells, enhanced sensitivity to ionizing radiation was observed for G6PD(-) cells exposed to single-dose or fractionated radiation. Fitting the single-dose radiation response data to the linear-quadratic model of radiation-induced cytotoxicity, we found that the G6PD(-) cells exhibited a significant enhancement in the alpha component of radiation-induced cell killing, while the values obtained for the beta component were similar in both the G6PD(-) and wild-type CHO cell lines. Here we report that the enhanced alpha component of radiation-induced cell killing is associated with a significant increase in the incidence of ionizing radiation-induced apoptosis in the G6PD(-) cells. These data suggest that G6PD and the oxidative pentose phosphate shunt protect cells from ionizing radiation-induced cell killing by limiting the incidence of radiation-induced apoptosis. The sensitivity to radiation-induced apoptosis was lost when the cDNA for wild-type G6PD was transfected into the G6PD(-) cell lines. Depleting GSH with l-BSO enhanced apoptosis of K1 cells while having no effect in the G6PD(-) cell line

Curcumin, a plant polyphenol, is a widely studied chemopreventive agent with demonstrated antitumor activities in preclinical studies and low toxicity profiles in multiple clinical trials against human malignancies. We previously showed that curcumin radiosensitizes cervical tumor cells without increasing the cytotoxic effects of radiation on normal human fibroblasts. Here we report that an inhibitory activity of curcumin on the antioxidant enzyme thioredoxin reductase-1 (TxnRd1) is required for curcumin-mediated radiosensitization of squamous carcinoma cells. Stable knockdown of TxnRd1 in both HeLa and FaDu cells nearly abolished curcumin-mediated radiosensitization. TxnRd1 knockdown cells showed decreased radiation-induced reactive oxygen species and sustained extracellular signal-regulated kinase 1/2 activation, which we previously showed was required for curcumin-mediated radiosensitization. Conversely, overexpressing catalytically active TxnRd1 in HEK293 cells, with low basal levels of TxnRd1, increased their sensitivity to curcumin alone and to the combination of curcumin and ionizing radiation. These results show the critical role of TxnRd1 in curcuminmediated radiosensitization and suggest that TxnRd1 levels in tumors could have clinical value as a predictor of response to curcumin and radiotherapy.

No abstract

The oxidative pentose phosphate cycle (OPPC) is necessary to maintain cellular reducing capacity during periods of increased oxidative stress. Metabolic flux through the OPPC increases stoichiometrically in response to a broad range of chemical oxidants, including those that generate reactive oxygen species (ROS). Here we show that OPPC sensitivity is sufficient to detect low levels of ROS produced metabolically as a function of the percentage of O 2 . We observe a significant decrease in OPPC activity in cells incubated under severe and moderate hypoxia (ranging from <0.01 to 4% O 2 ), whereas hyperoxia (95% O 2 ) results in a significant increase in OPPC activity. These data indicate that metabolic ROS production is directly dependent on oxygen concentration. Moreover, we have found no evidence to suggest that ROS, produced by mitochondria, are needed to stabilize hypoxia-inducible factor 1␣ (HIF-1␣) under moderate hypoxia. Myxothiazol, an inhibitor of mitochondrial electron transfer, did not prevent HIF-1␣ stabilization under moderate hypoxia. Moreover, the levels of HIF-1␣ that we observed after exposure to moderate hypoxia were comparable between 0 cells, which lack functional mitochondria, and the wild-type cells. Finally, we find no evidence for stabilization of HIF-1␣ in response to the non-toxic levels of H 2 O 2 generated by the enzyme glucose oxidase. Therefore, we conclude that the oxygen dependence of the prolyl hydroxylase reaction is sufficient to mediate HIF-1␣ stability under moderate as well as severe hypoxia.The primary role of the oxidative pentose phosphate cycle (OPPC) 2 in mammalian cells is to maintain the [NADPH/ NADPϩ] ratio, thereby helping to regulate the cellular redox equilibrium (1-3). Glucose-6-phosphate dehydrogenase (G6PD), the initial and rate-limiting enzyme of the OPPC, exists in a dimer-tetramer equilibrium with the tetramer being the catalytically active conformation. Each of four identical G6PD monomers contains a structural NADPϩ binding site (4, 5). When NADPϩ is bound at this site, formation of the active tetramer is favored. In non-stressed cells, the [NADPH]/ [NADPϩ] ratio is very high (approaching 1000) and flux through the OPPC is minimal. However, even a slight increase in [NADPϩ] can increase the number of active G6PD tetramers. Therefore, G6PD activity, by regulating flux through the OPPC, is uniquely sensitive to reactive oxygen species (ROS) as well as other chemical oxidants.The importance of the OPPC for the cellular response to ROS is evident from the elevated incidence of apoptosis that we observed in G6PD Ϫ Chinese hamster ovary cells following exposure to ionizing radiation (2). Likewise, Efferth et al. (6) observed an elevated incidence of oxidant-induced apoptosis in macrophages isolated from patients suffering from G6PD deficiency syndrome, while Fico et al. (42) observed H 2 O 2 -induced apoptosis in G6PD Ϫ mouse embryo fibroblasts. Notably, a 10-fold reduction in cloning efficiency was seen in G6PD Ϫ mouse embryo fibroblasts (MEFs) incubated in a...