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.

Incidental cervical metastases from thyroid carcinoma during neck dissection.

OBJECTIVES: To quantify and discuss the prevalence of unsuspected thyroid lymph node metastases discovered in specimens from neck dissection for head and neck squamous cell carcinoma (HNSCC) and discuss the impact on patient management. STUDY DESIGN: Retrospective study between May 2004 and January 2007. SETTING: University hospital. METHODS: Pathological analysis of cervical lymph node dissection performed during surgery for HNSCC in a total of 349 neck dissections in 266 consecutive patients. RESULTS: Twenty-one patients showed metastatic lymph nodes from thyroid cancer (prevalence 7.9%): 13 cases were metastatic from a papillary thyroid carcinoma and 8 cases from a follicular carcinoma. In 5 of the 21 patients, classical dissection was associated to recurrent nerve dissection and unilateral lobectomy; no thyroid carcinoma was found. Thirteen patients received radiotherapy for HNSCC. Follow-up comprised annual ultrasonographic examination of the neck and thyroid in these 21 patients. Total thyroidectomy was decided on in 5, with discovery of 3 micro-papillary thyroid carcinomas, in a single patient (complementary 131I treatment). No thyroid carcinomas were found for the other 4 patients. No patients died from thyroid carcinoma during follow-up (mean: 41 months). CONCLUSION: The prevalence of lymph node metastasis from thyroid carcinoma in cervical lymph node dissection during treatment of HNSCC seems higher (7.9%) than rates reported in the literature (0.3 to 1.6%). This may be due to the histopathological methods employed. Management of patients should be discussed in the light of thyroid ultrasonography and prognosis of HNSCC.

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.

Fluvastatin synergistically improves the antiproliferative effect of everolimus on rat smooth muscle cells by altering p27Kip1/cyclin E expression.

Multiple intracellular signaling pathways stimulate quiescent smooth muscle cells (SMCs) to exit from G(0) and re-enter the cell cycle. Thus, a combination of two drugs with different mechanisms of action may represent a suitable approach to control SMC proliferation, a prominent feature of in-stent restenosis. In the present study, we investigated the effect of everolimus, a mammalian target of rapamycin inhibitor, in combination with fluvastatin, a 3-hydroxy-3-methylglutaryl coenzyme A reductase inhibitor, on proliferation of rat SMCs. The antiproliferative action of everolimus was amplified by 2.5-fold by the addition of subliminal concentrations of fluvastatin (5 x 10(-7) M), lowering the IC(50) value from 2.5 x 10(-9) to 1.0 x 10(-9) M. The increased antiproliferative effect of everolimus by fluvastatin was prevented in the presence of mevalonate, farnesol, or geranylgeraniol, suggesting the involvement of prenylated proteins. Cell cycle analysis and [3H]thymidine incorporation assay demonstrated that the two drugs synergistically interfered with the progression of G(1) phase. In particular, the drug combination significantly up-regulated p27(Kip1) levels by 47.0%, suppressed cyclin E by 43.0%, and it reduced retinoblastoma (Rb) hyperphosphorylation by 79.0%, compared with everolimus alone. Retroviral overexpression of cyclin E conferred a significant resistance of rat SMCs to the antiproliferative action of the drug combination, measured by cell counting, [3H]thymidine incorporation, and cell cycle analysis, with higher levels of hyperphosphorylated form of Rb. Taken together, these results demonstrated that everolimus acts synergistically with fluvastatin to inhibit SMC proliferation by altering the expression of cyclin E and p27(kip1), which affects Rb phosphorylation and leads to G(1) phase arrest.

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.

SDH mutations in patients affected by paraganglioma syndromes: a personal experience.

Mutations in genes encoding mitochondrial succinate dehydrogenase (SDH) are frequently involved in the development of neural crest-derived (NCD) tumors, such as pheochromocytomas (PHEOs) or paragangliomas (PGLs). In this study we report the results of sequencing analysis in leukocyte DNA of patients affected by PHEO/PGL who turned out to be SDH mutation carriers. A nonsense germline heterozygous mutation (Q109X) was found in the exon 4 of the SDHD gene in the index cases of six unrelated families affected by PHEO/PGL. Haplotype analysis showed the presence of a founder effect. Affected patients showed high clinical variability, ranging from monolateral to bilateral glomus tumors, variably associated or not with PGLs or PHEOs. A novel missense SDHD variant, T112I, was also found in one of our families. A new missense G106D mutation, involving a highly conserved amino acid, was found in two sisters affected by bilateral glomus tumors. A P81L mutation associated with abdominal and head and neck PGL was detected in three families. A G12S variant of the SDHD gene was found in one patient affected by a PHEO. The finding of this variant in 3 of 100 control subjects suggests that it is a polymorphism and not a mutation. A novel IVS2-1G>T variant was found at intron 2 of SDHD gene in one patient affected by a glomus tumor. All the tumors associated with SDHD mutations were benign. Conversely, the only mutation we found in SDHB gene (IVS3+1G>A) was associated with a malignant PHEO.

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.

Doxorubicin overproduction in Streptomyces peucetius: cloning and characterization of the dnrU ketoreductase and dnrV genes and the doxA cytochrome P-450 hydroxylase gene.

Doxorubicin-overproducing strains of Streptomyces peucetius ATCC 29050 can be obtained through manipulation of the genes in the region of the doxorubicin (DXR) gene cluster that contains dpsH, the dpsG polyketide synthase gene, the putative dnrU ketoreductase gene, dnrV, and the doxA cytochrome P-450 gene. These five genes were characterized by sequence analysis, and the effects of replacing dnrU, dnrV, doxA, or dpsH with mutant alleles and of doxA overexpression on the production of the principal anthracycline metabolites of S. peucetius were studied. The exact roles of dpsH and dnrV could not be established, although dnrV is implicated in the enzymatic reactions catalyzed by DoxA, but dnrU appears to encode a ketoreductase specific for the C-13 carbonyl of daunorubicin (DNR) and DXR or their biosynthetic precursors. The highest DXR titers were obtained in a dnrX dnrU (N. Lomovskaya, Y. Doi-Katayama, S. Filippini, C. Nastro, L. Fonstein, M. Gallo, A. L. Colombo, and C. R. Hutchinson, J. Bacteriol. 180:2379-2386, 1998) double mutant and a dnrX dnrU dnrH (C. Scotti and C. R. Hutchinson, J. Bacteriol. 178:7316-7321, 1996) triple mutant. Overexpression of doxA in a doxA::aphII mutant resulted in the accumulation of DXR precursors instead of in a notable increase in DXR production. In contrast, overexpression of dnrV and doxA jointly in the dnrX dnrU double mutant or the dnrX dnrU dnrH triple mutant increased the DXR titer 36 to 86%.

Genetic co-inactivation of macrophage- and T-tropic HIV-1 chemokine coreceptors CCR-5 and CXCR-4 by intrakines.

CC-chemokine receptor (CCR)-5 is the principal coreceptor for the entry of macrophage (M)-tropic HIV-1 viruses into a cell, while CXC-chemokine receptor (CXCR)-4 is the principal coreceptor for T cell line (T)-tropic HIV-1. In this study, we utilized a novel intracellular chemokine (intrakine) strategy to co-inactivate genetically both CCR-5 and CXCR-4 in human lymphocytes. The principle of co-inactivation of CCR-5 and CXCR-4 was illustrated by targeting the CC-intrakine and CXC-intrakine to the lumen of the endoplasmic reticulum (ER) for intracellular blockade of the transport of newly synthesized chemokine coreceptors to the cell surface. The lymphocytes with the phenotypic knock-out of CCR-5 and CXCR-4 were found broadly to resist the infection of M-tropic, T-tropic and dual-tropic HIV-1 viruses. Moreover, the transduced lymphocytes retained normal cell features, including the responsiveness to mitogen and recall antigen stimulation. Thus, this study to our knowledge, is the first to demonstrate that genetic co-inactivation of the M- and T-tropic HIV-1 principal coreceptors in lymphocytes or other cells could be a viable strategy for the long-term control of HIV-1 infection.

Anti-HIV type 1 activity of wild-type and functional defective RANTES intrakine in primary human lymphocytes.

We have developed a genetic "intrakine" strategy to inactivate the CC-chemokine receptor 5 (CCR-5), the principal coreceptor for macrophage (M)-tropic HIV-1 viruses (Yang et al, 1997). The inactivation of CCR5 was achieved by targeting a modified CC-chemokine (RANTES) to the lumen of the endoplasmic reticulum (ER) to block the transport of the newly synthesized CCR-5. The transduced lymphocytes with the phenotypic CCR5 knockout were shown to be resistant to M-tropic HIV-1 infection. This study illustrated the feasibility of the intrakine strategy to block HIV-1 infection. In our current study, the potential clinical application of the intrakine approach was further evaluated in human peripheral blood lymphocytes (PBLs). PBLs were transduced with the RANTES intrakine gene by using retroviral vectors with the truncated low-affinity human nerve growth factor receptor (deltaNGFR) marker, and then isolated by an anti-NGFR antibody/magnetic bead method. The surface expression of CCR-5 in the transduced lymphocytes was dramatically inhibited, as demonstrated by flow cytometric assays. The transduced PBLs were shown to resist various types of M-tropic HIV-1 virus infection. The cell viability, cell proliferation rates, and cell surface markers of the intrakine-transduced PBLs were shown to be comparable to those of control PBLs. The transduced PBLs were also found to respond to the stimulation of various CXC- and CC-chemokines, other than RANTES. The transduced PBLs responded to tetanus antigen stimulation by increasing IL-2 production and cell proliferation. In addition, a functionally defective mutant of RANTES that retains its binding activity to CCR-5, but loses its signaling ability, was used to generate a mutant RANTES intrakine. The primary lymphocytes transduced with the mutant RANTES intrakine were found to be resistant to M-tropic HIV-1 infection. From these results, we conclude that the primary human lymphocytes transduced with either the wild-type or functionally defective RANTES intrakine are resistant to M-tropic HIV-1 infection, and maintain their basic biological functions. This study, therefore, indicates the potential clinical application of the intrakine approach for HIV-1 gene therapy.