A perylene derivative regulates HIF-1α and Stat3 signaling pathways.

It is becoming increasingly evident that improving the cure rate of many cancers will require treatment regimens hit more than one validated tumor targets. Developing an anti-cancer agent that targets two oncoproteins simultaneously is a promising strategy for accomplishing this goal. It would be expected to promote drug efficacy, reduce therapy-resistant without introducing additional toxic side effects. HIF-1α is a key regulator of the cellular response to hypoxia and is involved in tumor angiogenesis and cancer cell survival, glucose metabolism, and invasion. Stat3 has several oncogenic functions, including suppression of anti-tumor immune responses and promotion of inflammation. Recently, we have identified the perylene derivative, TEL03, as a dual inhibitor that targets both HIF-1α and Stat3. TEL03 blocks the expression of both HIF-1α and Stat3, regulated oncogenes (e.g., Bcl-2, VEGF, Glut1, and others) in cancer cells, and induces cancer cell apoptosis. The results demonstrated that: (i) TEL03 blocks Stat3 phosphorylation, and inhibits Stat3 transcriptional activity; and (ii) interferes the binding of HIF-1α to p300/CBP inducing its degradation by proteasomes under hypoxic conditions. Our in vivo tests showed that as a dual inhibitor, TEL03 dramatically inhibited tumor growth, and provided the evidence that targeting both HIF-1α and Stat3 simultaneously could be a promising strategy for breast and pancreatic cancer therapies.

Combined treatment targeting HIF-1α and Stat3 is a potent strategy for prostate cancer therapy.

BACKGROUND: The Stat3 pathway and the hypoxia-sensing pathway are both up-regulated in prostate cancer. Stat3 is a specific regulator of pro-carcinogenic inflammation and represents a promising therapeutic target. Hypoxia-inducible factor-1 (HIF-1)α, which mediates the cellular response to hypoxia, has been demonstrated to be over-expressed in many human cancers and is associated with poor prognosis and treatment failure in clinic. To develop a potent strategy to increase therapeutic efficacy and reduce drug resistance in prostate cancer therapy, we combined two anti-cancer agents: T40214 (a p-Stat3 inhibitor) and JG244 (a HIF-1α inhibitor) together to treat nude mice bearing human prostate tumor (DU145) and immunocompetent mice (C57BL/6) bearing murine prostate tumor (TRAMP-C2). METHODS: We employed in vitro and in vivo assays, including Western blots, cell cycle analysis, immunohistochemistry, TUNEL and xenograft models to determine the drug efficacy and mechanism of combination treatment of T40214 and JG244. RESULTS: We found that compared to treatment by T40214 or JG244 alone, the combination treatment using T40214 and JG244 together significantly suppressed growth of human or murine prostate tumors. Also, compared with apoptotic cells induced by T40214 or JG244 alone, the combined treatment greatly increased apoptosis in DU145 (P < 0.006) and TRAMP-C2 tumors (P < 0.008). CONCLUSIONS: Our results suggested that combination treatment including a HIF-1α/2α inhibitor not only has therapeutic efficacy in targeting HIF-1α/2α, but also could reduce the hypoxia-induced drug resistance to other therapies (e.g., T40214) and enhance drug efficacy. This approach could make prostate cancer treatments more effective.

G-rich oligonucleotides inhibit HIF-1alpha and HIF-2alpha and block tumor growth.

Hypoxia-inducible factor-1 (HIF-1) plays crucial roles in tumor promotion by upregulating its target genes, which are involved in energy metabolism, angiogenesis, cell survival, invasion, metastasis, and drug resistance. The HIF-1alpha subunit, which is regulated by O2-dependent hydroxylation, ubiquitination, and degradation, has been identified as an important molecular target for cancer therapy. We have rationally designed G-rich oligodeoxynucleotides (ODNs) as inhibitors of HIF-1alpha for human cancer therapy. The lead compounds, JG243 and JG244, which form an intramolecular parallel G-quartet structure, selectively target HIF-1alpha and decreased levels of both HIF-1alpha and HIF-2alpha (IC50 < 2 micromol/l) and also inhibited the expression of HIF-1-regulated proteins [vascular endothelial growth factor (VEGF), Bcl-2, and Bcl-XL], but did not disrupt the expression of p300, Stat3, or p53. JG-ODNs induced proteasomal degradation of HIF-1alpha and HIF-2alpha that was dependent on the hydroxylase activity of prolyl-4-hydroxylase-2. JG243 and JG244 dramatically suppressed the growth of prostate, breast, and pancreatic tumor xenografts. Western blots from tumor tissues showed that JG-ODNs significantly decreased HIF-1alpha and HIF-2alpha levels and blocked the expression of VEGF. The JG-ODNs are novel anticancer agents that suppress tumor growth by inhibiting HIF-1.

A G-quartet oligonucleotide blocks glycoprotein Ib-mediated platelet adhesion and aggregation under flow conditions.

Platelets arrest bleeding by adhering to and aggregating on the subendothelium exposed at the site of vessel injury. This process is initiated by the interaction between the subendothelium von Willebrand factor (VWF) and the glycoprotein (GP) Ib-IX-V complex on platelets. However, the same interaction also results in thrombosis at the site of a ruptured atherosclerotic plaque. Reagents regulating the GP Ib-VWF interaction will therefore have direct impact on haemostasis and thrombosis. We have characterised an oligonucleotide G-quartet (T30923) that specifically blocks VWF binding to GP Ibalpha, the VWF-binding subunit of the GP Ib-IX-V complex. We evaluated the potential interactions of T30923 with GP Ibalpha and VWF A1 domain by computer simulated molecular dockings, which identified four T30923 docking sites in the beta-sheets of the N-terminal region of GP Ibalpha (E14-D18, S39, D63-S64, and D83-S85). Experimentally, T30923 bound GP Ibalpha and dose-dependently blocked platelet aggregation induced by ristocetin and thrombin, but not by botrocetin, collagen, TRAP, and ADP. It also blocked shear-induced platelet aggregation and thrombus formation on immobilised VWF under arterial shear stress. These results demonstrate that T30923 may have therapeutic potentials to regulate the GP Ibalpha-VWF interaction.

T40214/PEI complex: a potent therapeutics for prostate cancer that targets STAT3 signaling.

BACKGROUND: Prostate cancer (PC) is the most common cancer among men in American and the second leading cause of cancer death. The treatment options employed for patients with advanced and metastatic PC are limited. As a critical mediator of oncogenic signaling, STAT3 is active in 82% of patients with PC. STAT3 has become a very important molecular target for PC therapy since it upregulates the oncogenes encoding apoptosis inhibitors, cell cycle regulators, and inducers of angiogenesis. However, no anti-tumor drug whose primary mode of action is to target STAT3 has yet reached the clinic. To this end, we have laid the initial groundwork to develop the STAT3-inhibiting G-quartet oligodeoxynucleotide (GQ-ODN), T40214, for treatment of PCs. METHODS: We employed in vitro and in vivo assays, including Western blots, EMSA, cell cycle analysis, TUNEL and xenograft models, to determine the drug efficacy and mechanism of T40214/PEI complex. RESULTS: The results demonstrated that (i) T40214 significantly inhibited STAT3 activation and induced apoptosis in both androgen-dependent and androgen-independent PC cells; (ii) T40214 delivered by ployethylenimine (PEI) significantly suppressed prostate tumor growth in tumor-bearing nude mice due to that T40214 inhibited STAT3 activation and then greatly promoted apoptosis, reduced angiogenesis and cell proliferation in prostate tumors. CONCLUSION: Our studies suggested that STAT3 is a critical oncogenic signal, which strongly influences the progression of PCs and that T40214/PEI complex is a promising candidate for treatment of patients with prostate tumors and represents a novel strategy for PC therapy.

High levels of circulating CD34 cells, dacrocytes, clonal hematopoiesis, and JAK2 mutation differentiate myelofibrosis with myeloid metaplasia from secondary myelofibrosis associated with pulmonary hypertension.

We studied 25 patients with myelofibrosis with myeloid metaplasia and 19 patients with secondary myelofibrosis associated with pulmonary hypertension (PH). In these 2 groups, we compared the peripheral-blood CD34 count, the clonality of granulocytes and platelets in peripheral blood, the mutational status of the JAK2 kinase gene, and the morphology of the peripheral blood and bone marrow. We found that the following were distinctive features of myelofibrosis with myeloid metaplasia but not of secondary myelofibrosis due to PH: high circulating CD34 cell count, the presence of clonal platelets and granulocytes and of peripheral-blood dacrocytes, and a JAK2 1849G>T (V617F) mutation. We conclude that these are intrinsic features of clonal progenitors present in patients with myelofibrosis due to myeloproliferative disorders and that these features are not due to the abnormal marrow architecture seen in secondary myelofibrosis.

Heterogeneity of the molecular biology of methemoglobinemia: a study of eight consecutive patients.

Congenital methemoglobinemia can be caused by mutations involving five different genes. We studied the etiology and molecular biology of eight consecutive patients with methemoglobinemia. Four had b5R mutations; two were novel. A novel intronic mutation caused markedly reduced mRNA resulting in type II methemoglobinemia. Three patients had acquired methemoglobinemia without any b5R mutations.

Congenital polycythemia with homozygous and heterozygous mutations of von Hippel-Lindau gene: five new Caucasian patients.

We report on five Caucasian patients with congenital polycythemia and mutations of the von Hipple-Lindau (VHL) gene: a compound heterozygote for the novel exon 1 (VHL 235C->T) and previously reported VHL 562C->G mutations; three homozygotes for Chuvash VHL 598C->T mutation; and a heterozygote for VHL 523->G mutation who also has ataxia-telangiectasia; a rare autosomal disease of childhood onset.

Identification of a human mutation of DMT1 in a patient with microcytic anemia and iron overload.

Divalent metal transporter 1 (DMT1) is a transmembrane protein crucial for duodenal iron absorption and erythroid iron transport. DMT1 function has been elucidated largely in studies of the mk mouse and the Belgrade rat, which have an identical single nucleotide mutation of this gene that affects protein processing, stability, and function. These animals exhibit hypochromic microcytic anemia due to impaired intestinal iron absorption, and defective iron utilization in red cell precursors. We report here the first human mutation of DMT1 identified in a female with severe hypochromic microcytic anemia and iron overload. This homozygous mutation in the ultimate nucleotide of exon 12 codes for a conservative E399D amino acid substitution; however, its pre-dominant effect is preferential skipping of exon 12 during processing of pre-messenger RNA (mRNA). The lack of full-length mRNA would predict deficient iron absorption in the intestine and deficient iron utilization in erythroid precursors; however, unlike the animal models of DMT1 mutation, the patient is iron overloaded. This does not appear to be due to up-regulation of total DMT1 mRNA. DMT1 protein is easily detectable by immunoblotting in the patient's duodenum, but it is unclear whether the protein is properly processed or targeted.

Instability of PRV-1 mRNA: a factor to be considered in PRV-1 quantification for the diagnosis of polycythemia vera.

High expression of PRV-1 mRNA in granulocytes has been proposed as a new diagnostic marker for polycythemia vera. We used real-time reverse transcription polymerase chain reaction (RT-PCR) to measure the levels of PRV-1 mRNA, GAPDH mRNA and 18S rRNA in granulocytes obtained from blood samples processed 2, 24 and 48 hours after collection and observed a significant decrease of PRV-1 levels after 24 and 48 hours. The instability of PRV-1 mRNA may affect the diagnostic value of the PRV-1 test in blood samples stored for extended periods.

The worldwide distribution of the VHL 598C>T mutation indicates a single founding event.

The first congenital defect of hypoxia-sensing homozygosity for VHL 598C>T mutation was recently identified in Chuvash polycythemia. Subsequently, we found this mutation in 11 unrelated individuals of diverse ethnic backgrounds. To address the question of whether the VHL 598C>T substitution occurred in a single founder or resulted from recurrent mutational events in human evolution, we performed haplotype analysis of 8 polymorphic markers covering 340 kb spanning the VHL gene on 101 subjects bearing the VHL 598C>T mutation, including 72 homozygotes (61 Chuvash and 11 non-Chuvash) and 29 heterozygotes (11 Chuvash and 18 non-Chuvash), and 447 healthy unrelated individuals from Chuvash and other ethnic groups. The differences in allele frequencies for each of the 8 markers between 447 healthy controls (598C) and 101 subjects bearing the 598T allele (P < 10(-7)) showed strong linkage disequilibrium. Haplotype analysis indicated a founder effect. We conclude that the VHL 598C>T mutation, the most common defect of congenital polycythemia yet found, was spread from a single founder 1,000 to 62,000 years ago.

Mutations of von Hippel-Lindau tumor-suppressor gene and congenital polycythemia.

The von Hippel-Lindau (pVHL) protein plays an important role in hypoxia sensing. It binds to the hydroxylated hypoxia-inducible factor 1 alpha (HIF-1 alpha) and serves as a recognition component of an E3-ubiquitin ligase complex. In hypoxia or secondary to a mutated VHL gene, the nondegraded HIF-1 alpha forms a heterodimer with HIF-beta and leads to increased transcription of hypoxia-inducible genes, including erythropoietin (EPO). The autosomal dominant cancer-predisposition von Hippel-Lindau (VHL) syndrome is due to inheritance of a single mutated allele of VHL. In contrast, we recently showed that homozygous germline 598C-->T VHL mutation leads to Chuvash polycythemia (CP). We subsequently found VHL mutations in three unrelated individuals unaffected with CP, one of whom was compound heterozygous for the 598C-->T mutation and another VHL mutation. We now report seven additional polycythemic patients with VHL mutations in both alleles. Two Danish siblings and another American boy were homozygous for the VHL 598C-->T mutation. Three unrelated white Americans were compound heterozygotes for 598C-->T and another VHL mutation, 562C-->G in two and 574C-->T in the third. Additionally, a Croatian boy was homozygous for a 571C-->G VHL mutation, the first example of homozygous VHL germline mutation causing polycythemia, other than the VHL 598C-->T mutation. We have not observed VHL syndrome-associated tumors in polycythemic subjects or their heterozygous relatives; however, this will need to be evaluated by longitudinal studies. Over all, we found that up to half of the consecutive patients with apparent congenital polycythemia and increased serum Epo we have examined have mutations of both VHL alleles. Those findings, along with reports of CP, underscore that VHL mutations are the most frequent cause of congenital polycythemia and define a new class of polycythemic disorder, polycythemias due to augmented hypoxia sensing.

Discrimination of polycythemias and thrombocytoses by novel, simple, accurate clonality assays and comparison with PRV-1 expression and BFU-E response to erythropoietin.

Essential thrombocythemia (ET) and polycythemia vera (PV) are clonal myeloproliferative disorders that are often difficult to distinguish from other causes of elevated blood cell counts. Assays that could reliably detect clonal hematopoiesis would therefore be extremely valuable for diagnosis. We previously reported 3 X-chromosome transcription-based clonality assays (TCAs) involving the G6PD, IDS, and MPP1 genes, which together were informative in about 65% of female subjects. To increase our ability to detect clonality, we developed simple TCA for detecting the transcripts of 2 additional X-chromosome genes: Bruton tyrosine kinase (BTK) and 4-and-a-half LIM domain 1 (FHL1). The combination of TCA established the presence or absence of clonal hematopoiesis in about 90% of female subjects. We show that both genes are subject to X-chromosome inactivation and are polymorphic in all major US ethnic groups. The 5 TCAs were used to examine clonality in 46 female patients along with assays for erythropoietin-independent erythroid colonies (EECs) and granulocyte PRV-1 mRNA levels to discriminate polycythemias and thrombocytoses. Of these, all 19 patients with familial polycythemia or thrombocytosis had polyclonal hematopoiesis, whereas 22 of 26 patients with clinical evidence of myeloproliferative disorder and 1 patient with clinically obscure polycythemia were clonal. Interestingly, interferon alpha therapy in 2 patients with PV was associated with reversion of clonal to polyclonal hematopoiesis. EECs were observed in 14 of 14 patients with PV and 4 of 12 with ET, and increased granulocyte PRV-1 mRNA levels were found in 9 of 13 patients with PV and 2 of 12 with ET. Thus, these novel clonality assays are useful in the diagnosis and follow-up of polycythemic conditions and disorders with increased platelet levels.

Mutations in the VHL gene in sporadic apparently congenital polycythemia.

The congenital polycythemic disorders with elevated erythropoietin (Epo) have been until recently an enigma, and abnormality in the hypoxia-sensing pathway has been hypothesized as a possible mechanism. The tumor suppressor von Hippel-Lindau (VHL) participates in the hypoxia-sensing pathway, as it binds to the proline-hydroxylated form of the hypoxia-inducible factor 1alpha (HIF-1alpha) and mediates its ubiquitination and proteosomal degradation. The loss of VHL function may result in the accumulation of HIF-1alpha and overproduction of HIF-1 downstream target genes including Epo. VHL syndrome is an autosomal dominant disorder predisposing to the development of tumors, due to inherited mutations in the VHL gene. Some rare patients with VHL syndrome have polycythemia, which has been attributed to Epo production by a tumor. It was recently found that homozygosity for the VHL Arg200Trp mutation is the cause of Chuvash polycythemia, an autosomal recessive polycythemic disorder characterized by elevated serum Epo and hypersensitivity of erythroid cells to Epo. We evaluated the role of VHL in 8 children with a history of polycythemia and an elevated serum Epo level and found 3 different germline VHL mutations in 4 of them. One child was homozygous for the Arg200Trp VHL mutation, and another compound heterozygous for the Arg200Trp and the Val130Leu mutations. Two children (siblings) were heterozygous for an Asp126Tyr mutation, one of them fulfilling some criteria of VHL syndrome. We propose that mutations of the VHL gene represent an important cause of pediatric sporadic polycythemias with an inappropriately high serum Epo concentration.

Disruption of oxygen homeostasis underlies congenital Chuvash polycythemia.

Chuvash polycythemia is an autosomal recessive disorder that is endemic to the mid-Volga River region. We previously mapped the locus associated with Chuvash polycythemia to chromosome 3p25. The gene associated with von Hippel-Lindau syndrome, VHL, maps to this region, and homozygosity with respect to a C-->T missense mutation in VHL, causing an arginine-to-tryptophan change at amino-acid residue 200 (Arg200Trp), was identified in all individuals affected with Chuvash polycythemia. The protein VHL modulates the ubiquitination and subsequent destruction of hypoxia-inducible factor 1, subunit alpha (HIF1alpha). Our data indicate that the Arg200Trp substitution impairs the interaction of VHL with HIF1alpha, reducing the rate of degradation of HIF1alpha and resulting in increased expression of downstream target genes including EPO (encoding erythropoietin), SLC2A1 (also known as GLUT1, encoding solute carrier family 2 (facilitated glucose transporter), member 1), TF (encoding transferrin), TFRC (encoding transferrin receptor (p90, CD71)) and VEGF (encoding vascular endothelial growth factor).

N-(4-hydroxyphenyl)retinamide (4-HPR) modulates GADD45 expression in radiosensitive bladder cancer cell lines.

We previously demonstrated that N-(4-hydroxyphenyl)retinamide (4-HPR) and gamma-irradiation, when used in combination, had a synergistic effect in inducing apoptosis in bladder cancer cells, suggesting that 4-HPR may increase radiosensitivity in bladder cancer cells. To unravel molecular correlates in this radiosensitizing effect of 4-HPR, we examined the baseline and 4-HPR-induced expression of GADD45 to elucidate possible mechanisms by which 4-HPR enhanced the effect of gamma-irradiation in three bladder cancer cell lines. To investigate the role of p53 in mediating the radiosensitizing effect of 4-HPR, we also examined mutations in exons 5-9 by using direct sequencing and the levels of p53 expression by using RT-PCR and Western blot, before and after treatment with 4-HPR in these bladder cancer cell lines. Two cell lines had low expression of GADD45, and a dose-dependent increase in GADD45 expression induced by 4-HPR was found in bladder cancer cell lines without p53 mutations in exons 5-9. A combination of gamma-irradiation and 4-HPR showed a significantly greater effect in enhancing GADD45 expression than either agent used alone. The results indicate that the combined treatment with 4-HPR and gamma-irradiation has a stronger effect on GADD45 expression than the treatment with either agent alone, which suggests that the two agents may have an additive/synergistic effect. However, a normal p53 function appears to be necessary for the dose-dependent induction of GADD45 by 4-HPR. Once our results are verified and replicated by other investigators, 4-HPR may have a potential clinical implication in effectively treating bladder cancer in combination with low-gamma-irradiation therapy.

Potassium-dependent folding: a key to intracellular delivery of G-quartet oligonucleotides as HIV inhibitors.

Several groups have demonstrated that G-rich oligonucleotides forming G-quartet structures display activity as potential drugs, such as potent HIV inhibitors. The delivery of G-quartet oligonucleotides to their intracellular targets is a key obstacle to overcome for their clinical success. Here we have developed a novel system to deliver G-rich oligonucleotides into the cell nucleus, e.g., the site of HIV integration. On the basis of the property of potassium-induced formation of G-quartet structure, we explored the difference of K(+) concentrations inside (140 mM) and outside (4 mM) cells to induce the G-rich oligonucleotides to form different structures inside and outside cells. The key steps of this delivery system include the following: (i) First, the G-quartet structure is denatured to form a lipid-DNA complex, so that the molecules can be well delivered into cells. (ii) Then the delivered molecules are induced to form G-quartet structures by potassium inside cells since the G-quartet structure is the primary requirement for inhibition of HIV-1 HIV integrase (IN) activity. The molecules of a novel G-quartet HIV inhibitor, T40214, with the sequence of (GGGC)(4) were successfully delivered into the nuclei of target cells, which significantly decreased HIV-1 replication and increased the probability to target HIV-1 IN in infected cells.

Acquired uniparental disomy of chromosome 9p is a frequent stem cell defect in polycythemia vera.

OBJECTIVE: Clonal stem cell proliferation and increased erythrocyte mass are hallmarks of the myeloproliferative disorder polycythemia vera (PV). The molecular basis of PV is unknown. METHODS: We carried out a genome-wide screening for loss of heterozygosity (LOH) and analyzed candidate genes within the LOH loci. RESULTS: Three genomic regions were identified on chromosomes 9p, 10q, and 11q. The presence of these LOHs in both myeloid and lymphoid cells indicated their stem cell origin. The 9pLOH prevalence is approximately 33% and is the most frequent chromosomal lesion described in PV so far. We report that the 9pLOH is due to mitotic recombination and therefore remains undetectable by cytogenetic analysis. Nineteen candidate genes were selected within the 9pLOH region for sequencing and expression analysis. No mutations were found in these genes; however, unexpectedly, increased expression of the transcription factor NFI-B was detected in granulocytes and CD34(+) cells in PV with 9pLOH. Since a member of the NFI gene family (NFI-X) was reported to result in TGF-beta resistance when overexpressed in vitro (TGF-beta is a known inhibitor of hematopoiesis), we transfected the NFI-B gene to the mouse 32D cell line. We found that overexpression of the NFI-B gene confers TGF-beta resistance in vitro. CONCLUSIONS: We characterized a new region on chromosome 9p frequently involved in LOH in PV. Analysis of genes within this 9pLOH region revealed increased expression of the NFI-B gene. Our in vitro studies suggest that TGF-beta resistance may be the physiologic mechanism of clonal stem cell expansion in PV.