Iron addiction: a novel therapeutic target in ovarian cancer.

Ovarian cancer is a lethal malignancy that has not seen a major therapeutic advance in over 30 years. We demonstrate that ovarian cancer exhibits a targetable alteration in iron metabolism. Ferroportin (FPN), the iron efflux pump, is decreased, and transferrin receptor (TFR1), the iron importer, is increased in tumor tissue from patients with high grade but not low grade serous ovarian cancer. A similar profile of decreased FPN and increased TFR1 is observed in a genetic model of ovarian cancer tumor-initiating cells (TICs). The net result of these changes is an accumulation of excess intracellular iron and an augmented dependence on iron for proliferation. A forced reduction in intracellular iron reduces the proliferation of ovarian cancer TICs in vitro, and inhibits both tumor growth and intraperitoneal dissemination of tumor cells in vivo. Mechanistic studies demonstrate that iron increases metastatic spread by facilitating invasion through expression of matrix metalloproteases and synthesis of interleukin 6 (IL-6). We show that the iron dependence of ovarian cancer TICs renders them exquisitely sensitive in vivo to agents that induce iron-dependent cell death (ferroptosis) as well as iron chelators, and thus creates a metabolic vulnerability that can be exploited therapeutically.

Effects of Anti-repulsive Guidance Molecule C (RGMc/Hemojuvelin) Antibody on Hepcidin and Iron in Mouse Liver and Tumor Xenografts.

OBJECTIVE: Hepcidin is a peptide hormone produced by the liver that regulates systemic iron homeostasis. Hepcidin is also synthesized by tumors, where it contributes to tumor growth by increasing the tumoral retention of iron. Targeted reduction of hepcidin may therefore be useful in reducing tumor growth. H5F9-AM8 is an antibody in preclinical development for the anemia of chronic disease that reduces hepcidin synthesis by binding to RGMc, a co-receptor involved in the transcriptional induction of hepcidin by BMP6. We explored the ability of H5F9-AM8 to act as an anti-tumor agent. METHODS: Effects of anti-hemojuvelin antibody on hepcidin synthesis were assessed by qRTPCR in tissue culture and in tumor xenografts and livers of mice treated with H5F9-AM8 or saline. Tumor growth was assessed using caliper measurements. Serum iron was measured colorimetrically and tissue iron was measured using western blotting and inductively coupled mass spectrometry. RESULTS: In tissue culture, the anti-hemojuvelin antibody H5F9-AM8 significantly reduced BMP6-stimulated hepcidin synthesis in HepG2 and other cancer cells. In mice, H5F9-AM8 reduced hepcidin in the liver and increased serum iron, total liver iron, and liver ferritin. Although hepcidin in tumors was also significantly decreased, H5F9-AM8 did not reduce tumor iron content, ferritin, or tumor growth. CONCLUSION: Anti-hemojuvelin antibody successfully reduces hepcidin in both tumors and livers but has different effects in these target organs: it reduces iron content and ferritin in the liver, but does not reduce iron content or ferritin in tumors, and does not inhibit tumor growth. These results suggest that despite their ability to induce hepcidin in tumors, the anti-tumor efficacy of systemic, non-targeted hepcidin antagonists may be limited by their ability to simultaneously elevate plasma iron. Tumor-specific hepcidin inhibitors may be required to overcome the limitations of drugs that target the synthesis of both systemic and tumor hepcidin.

Heat localization for targeted tumor treatment with nanoscale near-infrared radiation absorbers.

Focusing heat delivery while minimizing collateral damage to normal tissues is essential for successful nanoparticle-mediated laser-induced thermal cancer therapy. We present thermal maps obtained via magnetic resonance imaging characterizing laser heating of a phantom tissue containing a multiwalled carbon nanotube inclusion. The data demonstrate that heating continuously over tens of seconds leads to poor localization (∼ 0.5 cm) of the elevated temperature region. By contrast, for the same energy input, heat localization can be reduced to the millimeter rather than centimeter range by increasing the laser power and shortening the pulse duration. The experimental data can be well understood within a simple diffusive heat conduction model. Analysis of the model indicates that to achieve 1 mm or better resolution, heating pulses of ∼2 s or less need to be used with appropriately higher heating power. Modeling these data using a diffusive heat conduction analysis predicts parameters for optimal targeted delivery of heat for ablative therapy.

The core control system of intracellular iron homeostasis: a mathematical model.

Iron is a metal essential for cellular metabolism. However, excess iron available for reactions contributes to the formation of dangerous reactive oxygen species, such as the hydroxyl radical, via the Fenton reaction. Therefore, intracellular iron levels are tightly constrained by a control system of proteins. This paper contains a mathematical model, in the form of a system of five ordinary differential equations, of the core of this control system, including the labile iron pool as well as proteins that regulate uptake, storage, and export and are connected through negative feedback loops. The model is validated using data from an overexpression experiment with cultured human breast epithelial cells. The parameters in the mathematical model are not known for this particular cell culture system, so the analysis of the model was done for a generic choice of parameters. Through a mixture of analytical arguments and extensive simulations it is shown that for any choice of parameters the model reaches a unique stable steady state, thereby ruling out oscillatory behavior. It is shown furthermore that the model parameters are identifiable through suitable experiments.

Curcumin: from ancient medicine to current clinical trials.

Curcumin is the active ingredient in the traditional herbal remedy and dietary spice turmeric (Curcuma longa). Curcumin has a surprisingly wide range of beneficial properties, including anti-inflammatory, antioxidant, chemopreventive and chemotherapeutic activity. The pleiotropic activities of curcumin derive from its complex chemistry as well as its ability to influence multiple signaling pathways, including survival pathways such as those regulated by NF-kappaB, Akt, and growth factors; cytoprotective pathways dependent on Nrf2; and metastatic and angiogenic pathways. Curcumin is a free radical scavenger and hydrogen donor, and exhibits both pro- and antioxidant activity. It also binds metals, particularly iron and copper, and can function as an iron chelator. Curcumin is remarkably non-toxic and exhibits limited bioavailability. Curcumin exhibits great promise as a therapeutic agent, and is currently in human clinical trials for a variety of conditions, including multiple myeloma, pancreatic cancer, myelodysplastic syndromes, colon cancer, psoriasis and Alzheimer's disease.

p53-independent apoptosis mediated by tachpyridine, an anti-cancer iron chelator.

Iron is involved in essential biochemical reactions ranging from respiration to DNA synthesis. Consequently, iron deprivation has been proposed as a strategy for inhibition of tumor cell growth. We recently described a novel iron chelator, tachypyridine [N,N',N"-tris(2-pyridylmethyl)-cis,cis-1,3,5-triaminocyclohexane], and demonstrated that it not only inhibited growth of cultured tumor cells, but was actively cytotoxic. Here we explore the mechanisms underlying tachpyridine cytotoxicity. Using several criteria, including time-lapse video microscopy, DNA staining and TUNEL assays, tachpyridine was shown to specifically induce apoptotic cell death. Further, unlike numerous cytotoxic chemotherapeutic drugs which induce apoptosis by activating p53-dependent pathways, tachpyridine-mediated cell death did not require p53 activation. Although immunoblotting revealed rapid accumulation of p53 following treatment with tachpyridine, p21(WAF1) was not induced. Further, neither cytotoxicity nor apoptosis required p53. p53 null human lung cancer H1299 cells transfected with an ecdysone-inducible p53 exhibited equivalent sensitivity to tachpyridine in the presence and absence of p53, demonstrating the lack of requirement for p53 in an isogenic cell system. Further, time-lapse video microscopy and TUNEL assays demonstrated that both p53 null and p53 wild-type cells underwent apoptotic cell death in response to tachpyridine. In addition, in 55 human cancer cell lines the mean GI(50) of tachpyridine in cells with mutant p53 was virtually identical to the GI(50) in cells with wild-type p53. These results demonstrate that tachpyridine initiates an apoptotic mode of cell death that does not require functional p53. Since over 50% of human tumors contain a functionally defective p53 that reduces sensitivity to commonly used chemotherapeutic agents, such as etoposide and cisplatin, the ability of tachpyridine to induce apoptosis independently of p53 may offer an advantage in anti-tumor therapy.

Ferritin and the response to oxidative stress.

Iron is required for normal cell growth and proliferation. However, excess iron is potentially harmful, as it can catalyse the formation of toxic reactive oxygen species (ROS) via Fenton chemistry. For this reason, cells have evolved highly regulated mechanisms for controlling intracellular iron levels. Chief among these is the sequestration of iron in ferritin. Ferritin is a 24 subunit protein composed of two subunit types, termed H and L. The ferritin H subunit has a potent ferroxidase activity that catalyses the oxidation of ferrous iron, whereas ferritin L plays a role in iron nucleation and protein stability. In the present study we report that increased synthesis of both subunits of ferritin occurs in HeLa cells exposed to oxidative stress. An increase in the activity of iron responsive element binding proteins in response to oxidative stress was also observed. However, this activation was transient, allowing ferritin protein induction to subsequently proceed. To assess whether ferritin induction reduced the accumulation of ROS, and to test the relative contribution of ferritin H and L subunits in this process, we prepared stable transfectants that overexpressed either ferritin H or ferritin L cDNA under control of a tetracycline-responsive promoter. We observed that overexpression of either ferritin H or ferritin L reduced the accumulation of ROS in response to oxidant challenge.

Coordinate transcriptional and translational regulation of ferritin in response to oxidative stress.

The global increase in transcription of cytoprotective genes induced in response to oxidative challenge has been termed the antioxidant response. Ferritin serves as the major iron-binding protein in nonhematopoietic tissues, limiting the catalytic availability of iron for participation in oxygen radical generation. Here we demonstrate that ferritin is a participant in the antioxidant response through a genetically defined electrophile response element (EpRE). The EpRE of ferritin H identified in this report exhibits sequence similarity to EpRE motifs found in antioxidant response genes such as those encoding NAD(P)H:quinone reductase, glutathione S-transferase, and heme oxygenase. However, the EpRE of ferritin H is unusual in structure, comprising two bidirectional motifs arranged in opposing directions on complementary DNA strands. In addition to EpRE-mediated transcriptional activation, we demonstrate that ferritin is subject to time-dependent translational control through regulation of iron-regulatory proteins (IRP). Although IRP-1 is initially activated to its RNA binding (ferritin-repressing) state by oxidants, it rapidly returns to its basal state. This permits the translation of newly synthesized ferritin transcripts and ultimately leads to increased levels of ferritin protein synthesis following oxidant exposure. Taken together, these results clarify the complex transcriptional and translational regulatory mechanisms that contribute to ferritin regulation in response to prooxidant stress and establish a role for ferritin in the antioxidant response.

Adenovirus E1A blocks oxidant-dependent ferritin induction and sensitizes cells to pro-oxidant cytotoxicity.

Ferritin is a protein that oxidizes and sequesters intracellular iron in a mineral core. We have reported that the E1A oncogene selectively represses ferritin H transcription, resulting in reduced levels of the ferritin H protein. Here we demonstrate that cells respond to pro-oxidant challenge by inducing ferritin mRNA and protein, and that this response is completely blocked by E1A. Concordantly, E1A sensitized cells to the cytotoxic effects of oxidative stress and enhanced the accumulation of reactive oxygen species in response to pro-oxidant challenge. These results demonstrate that expression of E1A impedes the cellular response to oxidative stress, including the induction of ferritin.

Recent advances in the treatment of prostate cancer.

As new evidence for prostate cancer treatment has emerged in the last few years, longstanding controversies in the treatment of prostate cancer have resurfaced. A number of long-held tenets of prostate cancer therapy have been revisited, sometimes with surprising and challenging results. Although neoadjuvant hormonal therapy prior to radical prostatectomy decreases positive surgical margin rates, longer follow-up is needed to support survival improvement of this combined modality therapy. Androgen deprivation combined with radiation therapy appears to improve disease-free survival (and survival in one series) in patients with locally advanced cancer. Another approach to locally advanced prostate cancer using three-dimensional conformal radiation therapy may improve long term outcome. The data are currently insufficient to conclude that interstitial low dose rate brachytherapy is equivalent to conventional treatments: patients with small tumor volumes and low Gleason grade seem to obtain more benefit, whereas for large tumors with higher gleason grades this approach seems inferior to conventional treatments. In advanced prostate cancer recent data suggest that immediate hormonal therapy improves survival. In this group of patients the use of maximum androgen blockade remains controversial but may adversely affect quality of life compared to orchiectomy alone. Intermittent hormonal therapy may improve quality of life, although effect upon survival is unknown. Chemotherapy in combination with androgen deprivation is currently being studied as front-line therapy in advanced prostate cancer. Palliative benefit of chemotherapy for hormone refractory prostate cancer remains an important endpoint; survival advantage has not been seen in any randomized trials. Suramin may delay disease progression in hormone refractory prostate cancer. Many aspects of prostate cancer treatment will remain controversial until results of large, randomized trials with longer follow-up are available.

Transcriptional regulation of the mouse ferritin H gene. Involvement of p300/CBP adaptor proteins in FER-1 enhancer activity.

We previously identified a major enhancer of the mouse ferritin H gene (FER-1) that is central to repression of the ferritin H gene by the adenovirus E1A oncogene (Tsuji, Y., Akebi, N., Lam, T. K., Nakabeppu, Y., Torti, S. V., and Torti, F. M. (1995) Mol. Cell. Biol. 15, 5152-5164). To dissect the molecular mechanism of transcriptional regulation of ferritin H, E1A mutants were tested for their ability to repress FER-1 enhancer activity using cotransfection with ferritin H-chloramphenicol acetyltransferase (CAT) reporter constructs. Here we report that p300/CBP transcriptional adaptor proteins are involved in the regulation of ferritin H transcription through the FER-1 enhancer element. Thus, E1A mutants that failed to bind p300/CBP lost the ability to repress FER-1, whereas mutants of E1A that abrogated its interaction with Rb, p107, or p130 were fully functional in transcriptional repression. Transfection with E1A did not affect endogenous p300/CBP levels, suggesting that repression of FER-1 by E1A is not due to repression of p300/CBP synthesis, but to E1A and p300/CBP interaction. In addition, we have demonstrated that transfection of a p300 expression plasmid significantly activated ferritin H-CAT containing the FER-1 enhancer, but had a marginal effect on ferritin H-CAT with FER-1 deleted. Furthermore, both wild-type p300 and a p300 mutant that failed to bind E1A but retained an adaptor function restored FER-1 enhancer activity repressed by E1A. Sodium butyrate, an inhibitor of histone deacetylase, mimicked p300/CBP function in activation of ferritin H-CAT and elevation of endogenous ferritin H mRNA, suggesting that the histone acetyltransferase activity of p300/CBP or its associated proteins may contribute to the activation of ferritin H transcription. Recruitment of these broadly active transcriptional adaptor proteins for ferritin H synthesis may represent an important mechanism by which changes in iron metabolism are coordinated with other cellular responses mediated by p300/CBP.

Tumor cell cytotoxicity of a novel metal chelator.

We have synthesized a novel six-coordinate metal chelator from the triamine cis-1,3,5-triaminocyclohexane by the addition of a 2-pyridylmethyl pendant arm on each nitrogen, which we term tachpyr. The experiments described here were designed to explore whether this compound exhibits potential antitumor activity. When added to MBT2 or T24 cultured bladder cancer cells, tachpyr was profoundly cytotoxic, with an IC50 of approximately 4.6 micromol/L compared with 70 micromol/L for desferioxamine. To explore the mode of action of tachpyr, several metal complexes were prepared, including Fe(II), Ca(II), Mn(II), Mg(II), Cu(II), and Zn(II) tachpyr complexes. Of these, the Zn(II), Cu(II), and Fe(II) complexes were without toxic effect, whereas the Ca(II), Mn(II), and Mg(II) complexes remained cytotoxic. To further probe the role of Zn(II) and Cu(II) chelation in the cytotoxicity of tachpyr, sterically hindered tachpyr derivatives were prepared through N-alkylation of tachpyr. These derivatives were unable to strongly bind Fe(III) or Fe(II) but were able to bind Zn(II) and Cu(II). When added to cells, these sterically hindered tachpyr derivatives were nontoxic, consistent with a role of iron depletion in the cytotoxic mechanism of tachpyr. Further, the addition of tachpyr to proliferating cultures resulted in an early and selective inhibition of ferritin synthesis, an iron storage protein whose translation is critically dependent on intracellular iron pools. Taken together, these experiments suggest that tachpyr is a cytotoxic metal chelator that targets intracellular iron, and that the use of tachpyr in cancer therapy deserves further exploration.

Selective inhibition of muscle gene expression by oxidative stress in cardiac cells.

Reactive oxygen species have been suggested to play an important role in damage to cardiac tissue following ischemia and reperfusion. Oxygen radicals may also contribute to the cardiotoxicity of the anthracycline antibiotics, such as doxorubicin. We tested whether a selective inhibition of muscle gene expression, previously observed in cardiocytes treated with doxorubicin, might be reflective of a more generalized response evoked by oxidative stress in cardiac tissue. Cardiocytes in culture were exposed to hydrogen peroxide or glucose oxidase, and the effects on muscle gene expression were measured. Exposure to these agents led to a reduction in the levels of mRNA for the muscle-specific genes cardiac alpha-actin, troponin I, myosin light chain 2 (slow), and M isoform of creatine kinase, without affecting levels of the non-muscle genes pyruvate kinase and beta-actin. The magnitude of this effect was similar to that observed with doxorubicin. Although the hydrogen peroxide scavenging enzyme catalase and the intracellular radical scavengers N-acetylcysteine and 1,3-dimethyl-2-thiourea were without effect on doxorubicin-dependent reduction in gene expression, they inhibited the reduction in muscle gene expression mediated by hydrogen peroxide. These observations suggest that oxygen free radicals modulate muscle gene expression in cardiocytes by a pathway distinct from that utilized by doxorubicin.

Human H-kininogen is a ferritin-binding protein.

H-kininogen is a multifunctional protein: it inhibits cysteine proteases, plays a role in contact activation of the coagulation cascade, and is the precursor of the potent proinflammatory peptide bradykinin. In the experiments described here, we identify H-kininogen as a ferritin-binding protein. Ferritin is a cellular and serum protein that is elevated in acute and chronic inflammation and many cancers. Despite numerous reports of ferritin-binding protein(s) in human serum, the nature and function of these proteins remain unclear. As a first step in characterizing the interaction between ferritin and its binding protein(s), we devised a ligand blot assay and used it to guide purification of a ferritin-binding protein from human serum. Edman degradation of the purified protein determined the sequence HNLGHGHK(H)ERDQGHG, a sequence with identity to residues 421-436 of human H-kininogen. These results were confirmed by demonstrating that commercially purified H-kininogen possessed ferritin binding activity and that ferritin binding could not be detected in plasma from kininogen-deficient individuals. Ligand blot assays mapped the ferritin binding domain to the light chain of H-kininogen chain, and revealed that both H and L recombinant ferritins possess H-kininogen binding activity. The unexpected identification of H-kininogen as a ferritin-binding protein may link ferritin in the complex chain of interactions by which H-kininogen mediates its multiple effects in contact activation and inflammation.

Endocrine therapy of prostate cancer.

Endocrine therapy is effective treatment for patients with metastatic prostate cancer. Most patients will benefit from androgen withdrawal in terms of symptomatic relief and delay in progression of diseases. It does not, however, cure patients with metastatic prostate cancer. This finding emphasizes the need for the development of effective nonendocrine therapies.

Activation of the ferritin H enhancer, FER-1, by the cooperative action of members of the AP1 and Sp1 transcription factor families.

We have previously reported that the adenovirus E1A oncogene represses the transcription of the H subunit of the mouse ferritin gene. Subsequent analyses defined FER-1, a 37-nucleotide sequence located 4.1 kilobases proximal to the start site of transcription, as the target of E1A-mediated transcriptional repression and as an enhancer of the ferritin H gene. FER-1 is composed of an AP1-like sequence followed by an element with dyad symmetry. To achieve maximal enhancer activity and transcriptional repression by E1A, both elements were essential. Using gel retardation assays, we now demonstrate that the binding complex for the AP1-like sequence of FER-1 contains JunD, FosB, and ATF1. Furthermore, JunD and FosB were able to activate FER-1 enhancer activity by transient cotransfection with ferritin H-chloramphenicol acetyltransferase reporter constructs. This augmented enhancer activity was inhibited by E1A. In addition, we have defined the minimal sequence in the dyad element of FER-1 required for protein interaction. This was determined to be a C-rich sequence to which Sp1 and Sp3 bind. Experiments with recombinant proteins indicate that members of both transcription factor families simultaneously bind FER-1. Taken together, these results elucidate molecular mechanisms involved in the transcriptional regulation of a pivotal gene in iron metabolism and provide insights into the contribution of the Sp1 family to the activation of AP1-dependent enhancers.

Systemic chemotherapy of transitional cell carcinoma of the urothelium.

Although the incidence of bladder cancer has increased in recent years, survival has also improved. Chemotherapy has made a substantial impact on this disease and now is used in patients with advanced or metastatic disease as well as in select patients with high-risk muscle invasive disease. While cisplatin remains the most active single antineoplastic agent, several other agents including methotrexate, vinblastine, and Adriamycin (doxorubicin) have important activity. More recently, paclitaxel and gemcitabine have shown promising activity in bladder cancer. Multidrug combination therapy has provided more frequent and durable responses than single agent therapy. Regimens containing cisplatin and methotrexate have been shown to be most effective in the treatment of advanced disease. Adjuvant chemotherapy regimes typically have included cisplatin or cisplatin and methotrexate combinations. However, studies have been limited and further prospective trials are required to determine the role of adjuvant chemotherapy. Multiple studies have investigated neoadjuvant chemotherapy with cisplatin and methotrexate combinations or anthracycline-based regimens, but study results are mixed. Further trials will be required to define the role of neoadjuvant chemotherapy in bladder cancer.