Identification of the antineoplastic agent 6-mercaptopurine as an activator of the orphan nuclear hormone receptor Nurr1

The purine anti-metabolite 6-mercaptopurine is one of the most widely used drugs for the treatment of acute childhood leukemia and chronic myelocytic leukemia. Developed in the 1950s, the drug is also being used as a treatment for inflammatory diseases such as Crohn's disease. The antiproliferative mechanism of action of this drug and other purine anti-metabolites has been demonstrated to be through inhibition of de novo purine synthesis and incorporation into nucleic acids. Despite the extensive clinical use and study of 6-mercaptopurine and other purine analogues, the cellular effects of these compounds remain relatively unknown. More recently, purine anti-metabolites have been shown to function as protein kinase inhibitors and to regulate gene expression. In an attempt to find small molecule regulators of the orphan nuclear receptor Nurr1, interestingly, we identified 6-mercaptopurine as a specific activator of this receptor. A detailed analysis of 6-mercaptopurine regulation of Nurr1 demonstrates that 6-mercaptopurine regulates Nurr1 through a region in the amino terminus. This activity can be inhibited by components of the purine biosynthesis pathway. These findings indicate that Nurr1 may play a role in mediating some of the antiproliferative effects of 6-mercaptopurine and potentially implicate Nurr1 as a molecular target for treatment of leukemias.     

Pharmacologic circumvention of multidrug resistance

The ability of malignant cells to develop resistance to chemotherapeutic drugs is a major obstacle to the successful treatment of clinical tumors. The phenomenon multidrug resistance (MDR) in cancer cells results in cross-resistance to a broad range of structurally diverse antineoplastic agents, due to outward efflux of cytotoxic substrates by themdr1 gene product, P-glycoprotein (P-gp). Numerous pharmacologic agents have been identified which inhibit the efflux pump and modulate MDR. The biochemical, cellular and clinical pharmacology of agents used to circumvent MDR is analyzed in terms of their mechanism of action and potential clinical utility. MDR antagonists, termed chemosensitizers, may be grouped into several classes, and include calcium channel blockers, calmodulin antagonists, anthracycline andVinca alkaloid analogs, cyclosporines, dipyridamole, and other hydrophobic, cationic compounds. Structural features important for chemosensitizer activity have been identified, and a model for the interaction of these drugs with P-gp is proposed. Other possible cellular targets for the reversal of MDR are also discussed, such as protein kinase C. Strategies for the clinical modulation of MDR and trials combining chemosensitizers with chemotherapeutic drugs in humans are reviewed. Several novel approaches for the modulation of MDR are examined.Abbreviations ALL acute lymphocytic leukemia - AML acute myelogenous leukemia - CaM calmodulin - CsA cyclosporin A - MDR multidrug resistance - P-gp P-glycoprotein - PMA phorbol 12-myristate 13-acetate - PKC protein kinase C     

Reversed-phase high-performance liquid chromatographic assay method for quantitating 6-mercaptopurine and its methylated and non-methylated metabolites in a single sample

Methods of assaying 6-mercaptopurine (6MP) and its methylated and non-methylated metabolites are essential for the therapeutic dose in treating patients with acute lymphoblastic leukemia. However, previous methods are technically complicated and unsuitable for clinical use. Thus, we have now developed a method utilizing reversed-phase high-performance liquid chromatography (HPLC) in order to quantify these compounds in human red blood cells (RBCs) in a single sample to serve as an index of cytotoxic activity. The agents 6MP, 6-thioguanine (6TG) and 6-methylmercaptopurine (6MMP) were well separated by this assay. Linear relationships were observed between the peak areas and the RBC concentrations of 6MP, 6TG and 6MMP over the range of 20–2000, 18–1800 and 18–1800 pmol per 25 mg hemoglobin (Hb), respectively. The limit of quantitation of the assay is 20, 18 and 18 pmol per 25 mg Hb, respectively. This assay system is suitable for routine clinical use.     

Pharmacogenomics of acute lymphoblastic leukemia

Pharmacogenomics of acute lymphoblastic leukemia (ALL) evolved rapidly in the past few years. Majority of recent findings concerns knowledge on key components of ALL treatment, 6-mercaptopurine and methotrexate. Leukemia is the most common cancer affecting children, with ALL comprising 80 % of all leukemia cases. Introduction of treatment protocols composed of several chemotherapeutic agents improved importantly survival in patients with ALL. Nevertheless, ALL is still the leading cause of cancer-related death in children. Interindividual differences in drug responses are an important cause of resistance to treatment and adverse drug reactions. Identifying pharmacogenomic determinants of drugs used in ALL treatment may allow for prospective identification of patients with suboptimal drug responses allowing for complementation of traditional treatment protocols by genotype-based drug dose adjustment.     

Therapeutic potential of the chemokine receptor CXCR4 antagonists as multifunctional agents

The chemokine receptor CXCR4 possesses multiple critical functions in normal and pathologic physiology. CXCR4 is a G-protein-coupled receptor that transduces signals of its endogenous ligand, the chemokine CXCL12 (stromal cell-derived factor-1, SDF-1). The interaction between CXCL12 and CXCR4 plays an important role in the migration of progenitors during embryologic development of the cardiovascular, hemopoietic, central nervous systems, and so on. This interaction is also known to be involved in several intractable disease processes, including HIV infection, cancer cell metastasis, leukemia cell progression, rheumatoid arthritis (RA), and pulmonary fibrosis. It is conjectured that this interaction may be a critical therapeutic target in all of these diseases, and several CXCR4 antagonists have been proposed as potential drugs. Fourteen-mer peptides, T140 and its analogues, were previously developed in our laboratory as specific CXCR4 antagonists that were identified as HIV-entry inhibitors, anti-cancer-metastatic agents, anti-chronic lymphocytic/acute lymphoblastic leukemia agents, and anti-RA agents. Cyclic pentapeptides, such as FC131 [cyclo(D-Tyr-Arg-Arg-L-3-(2-naphthyl)alanine-Gly)], were also previously found as CXCR4 antagonist leads based on pharmacophores of T140. This review article describes the elucidation of multiple functions of CXCR4 antagonists and the development of a number of low-molecular weight CXCR4 antagonists involving FC131 analogues and other compounds with different scaffolds including linear-type structures.     

Dihydrofolate reductase as a therapeutic target

The folate antagonists are an important class of therapeutic compounds, as evidenced by their use as antiinfective, antineoplastic, and antiinflammatory drugs. Thus far, all of the clinically useful drugs of this class have been inhibitors of dihydrofolate reductase (DHFR), a key enzyme in the synthesis of thymidylate, and therefore, of DNA. The basis of the antiinfective selectivity of these compounds is clear; the antifolates trimethoprim and pyrimethamine are potent inhibitors of bacterial and protozoal DHFRs, respectively, but are only weak inhibitors of mammalian DHFRs. These species-selective agents apparently exploit the differences in the active site regions of the parasite and host enzymes. Methotrexate is the DHFR inhibitor used most often in a clinical setting as an anticancer drug and as an antiinflammatory and immunosuppressive agent. Considerable progress has been made recently in understanding the biochemical basis for the selectivity of this drug and the biochemical mechanism (or mechanisms) responsible for the development of resistance to treatment with the drug. This understanding has led to a new generation of DHFR inhibitors that are now in clinical trials.     

Involvement of the concentrative nucleoside transporter 3 and equilibrative nucleoside transporter 2 in the resistance of T-lymphoblastic cell lines to thiopurines

Mechanisms of resistance to thiopurines, 6-mercaptopurine (6-MP) and 6-thioguanine (6-TG) were investigated in human leukemia cell lines. We developed two 6-MP- and 6-TG-resistant cell lines from the human T-lymphoblastic cell line (MOLT-4) by prolonged exposure to these drugs. The resistant cells were highly cross resistant to 6-MP and 6-TG, and exhibited marked reduction in cellular uptake of 6-MP (70% and 80%, respectively). No significant modification of the activities of hypoxanthine-guanine phosphoribosyl transferase, thiopurine methyltransferase or inosine monophosphate dehydrogenase was observed. Real-time PCR of concentrative nucleoside transporter 3 (CNT3) and equilibrative nucleoside transporter 2 (ENT2) of resistant cells showed substantial reductions in expression of messenger RNAs. Small interfering RNA designed to silence the CNT3 and ENT2 genes down-regulated the expression of these genes in leukemia cells. These decreases were accompanied by reduction of transport of 6-MP (47% and 21%, respectively) as well as its cytocidal effect (30% and 21%, respectively). Taken together these results show that CNT3 and ENT2 play a key role in the transport of 6-MP and 6-TG by leukemia cells. From a clinical point of view determination of CNT3 and ENT2 levels in leukemia cells may be useful in predicting the efficacy of thiopurine treatment.     

Nonspecific inhibition of precursor uptake by alkyl-substituted purines and prednisolone

Of 43 compounds, encompassing a wide variety of biological activities, only the same six (9-cyclopentyladenine, 9-benzyladenine,9-n-butyladenine, 6-benzylthiopurine, 9-cyclopentyl-6-mercaptopurine, and prednisolone), at concentrations less than 10 times the toxic amounts, inhibited precursor incorporation into lipids, glycoproteins, and DNA of logarithmically-growing adenocarcinoma 755 and leukemia LI 210 cells in culture. For adenocarcinoma 755 cells, each of the six agents, added subsequent to choline, had little effect on the incorporation of choline into lipids, an indication that their action was on uptake of the precursor rather than on a bio-synthetic step. Direct measurements of choline and thymidine uptake into cells of both types showed that each of the six agents apparently blocked choline incorporation into lipids and thymidine incorporation into nucleic acids by inhibiting uptake of the precursors.     

Comprehensive screening of the thiopurine methyltransferase polymorphisms by denaturing high-performance liquid chromatography

The drug-metabolizing enzyme thiopurine S-methyltransferase (TPMT) catalyzes the S-methylation of thiopurines such as 6-mercaptopurine, 6-thioguanine, and azathiopurine, which are used as immunosuppressants and in the treatment of acute lymphoblastic leukemia and rheumatoid arthritis. TPMT enzymatic activity is a polymorphic trait, and poor metabolizers may develop life-threatening bone marrow failure. To avoid such adverse effects, the TPMT enzymatic activity in patients' red blood cells (RBCs) is routinely measured prior to thiopurine administration in a limited number of oncology clinics. In the present study, we took advantage of a highly sensitive and specific automated denaturing high-performance liquid chromatography (dHPLC) technique that not only detects known polymorphic alleles, but also identifies previously uncharacterized sequence variants. We developed a dHPLC-based protocol to analyze the entire coding region and validated the protocol to detect all 16 previously described variant alleles. We further analyzed the entire coding region of the TPMT gene in 288 control samples collected worldwide and identified two novel amino acid substitutions Arg163Cys (487C>T) and Arg226Gln (677G>A) within exons 7 and 10, respectively. The clinical application of this comprehensive screening system for examining the entire TPMT gene would help to identify patients at risk for bone marrow failure prior to 6-mercaptopurine therapy.     

Aminomethylpiperazines as selective urotensin antagonists

Aminomethylpiperazines, reported previously as being kappa-opioid receptor agonists, were identified as lead compounds in the development of selective urotensin receptor antagonists. Optimized substitution of the piperazine moiety has provided high affinity urotensin receptor antagonists with greater than 100-fold selectivity over the kappa-opioid receptor. Select compounds were found to inhibit urotensin-induced vasoconstriction in isolated rat aortic rings consistent with the hypothesis that an urotensin antagonist may be useful for the treatment of hypertension.     

Oblimersen Bcl-2 antisense: facilitating apoptosis in anticancer treatment

The components of the apoptotic program are targets for anticancer therapy. Bcl-2 protein inhibits apoptosis and confers resistance to treatment with traditional cytotoxic chemotherapy, radiotherapy, and monoclonal antibodies (mAb). Oblimersen sodium (G3139, Genasense, Genta Inc., Berkeley Heights, NJ) is an antisense oligonucleotide (AS-ON) compound designed to specifically bind to the first 6 codons of the human bcl-2 mRNA sequence, resulting in degradation of bcl-2 mRNA and subsequent decrease in Bcl-2 protein translation. Oblimersen is the first oligonucleotide to demonstrate proof of principle of an antisense effect in human tumors by the documented downregulation of the target Bcl-2 protein. A growing body of preclinical and clinical evidence suggests that oblimersen synergizes with many cytotoxic and biologic/immunotherapeutic agents against a variety of hematologic malignancies and solid tumors. Randomized clinical trials are currently underway to evaluate the efficacy and tolerability of oblimersen in combination with cytotoxic chemotherapy in chronic lymphocytic leukemia, multiple myeloma, malignant melanoma, and non-small cell lung cancer. In addition, nonrandomized trials are under way to evaluate oblimersen in non-Hodgkin's lymphoma, acute myeloid leukemia, and hormone-refractory prostate cancer. Preclinical data also support the clinical evaluation of oblimersen in additional tumor types, including chronic myelogenous leukemia and breast, small cell lung, gastric, colon, bladder, and Merkel cell cancers. Enhancement of the efficacy of anticancer treatments with oblimersen Bcl-2 antisense therapy represents a promising new apoptosis-modulating strategy, and ongoing clinical trials will test this therapeutic approach.     

Detection of minimal residual disease in acute leukemia by flow cytometry.

Patients with acute leukemia in clinical remission may still have up to 10(10) residual malignant cells (the upper limit of detection by standard morphologic techniques). Sensitive techniques to detect minimal residual disease (MRD) may allow better estimates of the leukemia burden and help the selection of appropriate therapeutic strategies. Flow cytometry and polymerase chain reaction have emerged as the most promising methods for detecting submicrospopic levels of leukemia. Flowcytometric detection of MRD is based on the identification of immunophenotypic combinations expressed on leukemic cells but not on normal hematopoietic cells. It affords the detection of one leukemic cell among 10,000 normal bone marrow cells, and can be currently applied to at least two thirds of all patients with acute leukemia. Prospective studies in large series of patients have demonstrated a strong correlation between MRD levels during clinical remission and treatment outcome. Therefore, MRD assays can be reliably used to assess early response to treatment and predict relapse. In this review, we discuss methodologic aspects and clinical results of flowcytometric detection of MRD in patients with acute leukemia.     

Monoclonal antibodies to carbohydrate antigens in autologous bone marrow transplantation

Normal and malignant myeloid cells express a highly immunogenic oligosaccharide, lacto-n-fucopentaose-III (LNF-III), that has been identified by numerous monoclonal antibodies (MoAb). We have been interested in the use of a particular monoclonal antibody to LNF-III, PM-81, in the treatment of patients with acute myelogenous leukemia using the antibody to treat bone marrow in vitro. Following in vitro treatment of bone marrow with PM-81 and another MoAb, AML-2-23, the remaining cells are used as an autograft in a patient treated with high-dose chemotherapy and radiotherapy. In order to enhance the ability of the MoAb to lyse leukemic cells in the remission bone marrow, we have explored the effect of neuraminidase treatment on leukemia cells. In this paper we describe that myeloid leukemia cells expressing low levels of LNF-III by immunofluorescence can be shown to have high levels of LNF-III after neuraminidase treatment. In addition, we show that normal bone marrow progenitor cells do not have cryptic LNF-III antigen, thus allowing the application of this finding to the clinical setting. Moreover, we have shown that leukemia colony-forming cells from one patient with acute myelogenous leukemia express cryptic LNF-III and that after exposure to neuraminidase there was an increased ability of PM-81 in the presence of complement to eliminate these colony forming cells. These data indicate that the LNF-III moiety is almost universally expressed on myeloid leukemia cells and their progenitors but not expressed on normal progenitors. Thus, it may be possible to enhance leukemia cell kill in vitro by neuraminidase treatment of bone marrow.     

Synthesis and properties of poly(9-vinylpurine-6-thiol), a polyvinyl thiopurine analog of polyriboinosinic acid

The chemical synthesis and some of the properties of poly(9-vinylpurine-6-thiol), a polyvinyl derivative of 6-mercaptopurine, are described. Analogous to some other polyvinyl compounds, this polymer interacts with polyribonucleotides by formation of double-stranded complexes. Its antiviral and cytostatic properties are currently under investigation.     

The inhibitor of apoptosis protein family and its antagonists in acute leukemias

The Inhibitor of Apoptosis Protein family (IAP) functions as inhibitors of apoptotic pathways, both death receptor- and mitochondrial mediated. We detail the current body of knowledge for the IAP family with regard to their structure and function, their expression in normal and leukemic cells, and their prognostic importance in acute leukemia. Although there is some evidence that IAPs play an important role in the chemoresistance of leukemia cell lines, little is known about their influence on this phenomenon in acute leukemia cells of human origin. IAPs are also explored as a specific target for new antitumor strategies, including antisense oligonucleotides of XIAP (X-chromosome-linked IAP) or survivin and small molecules of polyphenylurea-based XIAP inhibitors. Several proteins negatively regulate the function of the IAP family. One of those antagonists is Smac/DIABLO. Short peptides of Smac were found to enhanced apoptosis, induced by chemo- or immunotherapy, in the leukemic cells in vitro. Moreover, small-molecule agents, resembling Smac/DIABLO in function, were shown to potentiate cytotoxicity of chemotherapy in different malignancies. IAPs, exhibiting downstream influence on both external and intrinsic pathways as well as on some caspase-independent mechanisms of apoptosis, are potentially attractive target for anti-tumor therapy, although their role in the pathology and prognosis of acute leukemia has to be further elucidated.     

抗凋亡蛋白Mcl-1及其抑制剂的研究进展

骨髓细胞白血病蛋白Mcl-1是Bcl-2家族蛋白中重要的抗凋亡蛋白成员,其在多种恶性肿瘤(急性细胞性白血病、多发性骨髓瘤等)中都具有高表达的特点,导致肿瘤细胞对传统化疗药物及Bcl-2抑制剂产生耐药性。Mcl-1作为抗肿瘤药物研发的重要靶点正日益受到相关研究人员的关注,其中Mcl-1新型抑制剂以及联合抑制剂的研究取得了较大进展。本文将对Mcl-1蛋白结构和功能以及相关抑制剂的研究做更深入的分析和总结。     

The Management of Acute Leukemia

The therapy of acute leukemia has improved rapidly in the last two decades. Using available therapeutic agents, complete clinical and hematological remission can be achieved regularly in children with acute lymphocytic leukemia. The choice of chemotherapeutic agent, management of complications of hemorrhage and infection, and recognition of prognostic factors are important for the induction of a hematological remission. While patients in complete hematological remission are free of evidence of disease they still have residual leukemic cells, but in our present state of knowledge and with available techniques, we are unable to detect these. For this reason it is important to treat patients while in remission. The importance of dosage schedule for remission maintenance chemotherapy is stressed.In patients studied to date, regardless of the treatment used, the disease has recurred eventually. Available therapeutic agents are highly effective and highly selective, but they still fall short of providing ideal control of the disease. The continuing search for new chemotherapeutic agents is aided by the knowledge gained and techniques developed with current agents.     

Inhibition of monoamine oxidase B by selective adenosine A2A receptor antagonists

Adenosine receptor antagonists that are selective for the A(2A) receptor subtype (A(2A) antagonists) are under investigation as possible therapeutic agents for the symptomatic treatment of the motor deficits associated with Parkinson's disease (PD). Results of recent studies in the MPTP mouse model of PD suggest that A(2A) antagonists may possess neuroprotective properties. Since monoamine oxidase B (MAO-B) inhibitors also enhance motor function and reduce MPTP neurotoxicity, we have examined the MAO-B inhibiting properties of several A(2A) antagonists and structurally related compounds in an effort to determine if inhibition of MAO-B may contribute to the observed neuroprotection. The results of these studies have established that all of the (E)-8-styrylxanthinyl derived A(2A) antagonists examined display significant MAO-B inhibitory properties in vitro with K(i) values in the low micro M to nM range. Included in this series is (E)-1,3-diethyl-8-(3,4-dimethoxystyryl)-7-methylxanthine (KW-6002), a potent A(2A) antagonist and neuroprotective agent that is in clinical trials. The results of these studies suggest that MAO-B inhibition may contribute to the neuroprotective potential of A(2A) receptor antagonists such as KW-6002 and open the possibility of designing dual targeting drugs that may have enhanced therapeutic potential in the treatment of PD.     

Human recombinant IL-6: clinical promise.

The biology of interleukin-6 (IL-6) has been extensively reviewed. This article therefore does not repeat in detail how this molecule was discovered and its cDNA cloned, or the molecular mechanisms of its synthesis, its structural features, and its basic biological functions. Instead, three questions are addressed more closely: (i) How is IL-6 involved in various disease states? (ii) Can IL-6 be useful as a diagnostic clinical marker? (iii) Are there good reasons to expect that IL-6, IL-6 derivatives or IL-6 antagonists may eventually be utilized as therapeutic agents?     

Determination of extracellular and intracellular thiopurines and methylthiopurines by high-performance liquid chromatography

The thiopurine antimetabolites 6-thioguanine and 6-mercaptopurine are important chemotherapeutic drugs in the treatment of childhood acute lymphoblastic leukaemia. Measurement of metabolites of these thiopurines is important because correlations exist between levels of these metabolites and the prognosis in childhood acute lymphoblastic leukemia. The reversed-phase method for the determination of extracellular thiopurine nucleosides and bases was previously developed and has been modified such that methylthiopurine nucleosides, bases, thioxanthine and thiouric acid can be measured also. The anion-exchange method enables the determination of intracellular mono-, di- and triphosphate (methyl)thiopurine nucleotides in one run. Extraction on ice with perchloric acid and dipotassium hydrogenphosphate results in good recoveries for (methyl)thiopurine nucleotides in lymphoblasts and peripheral mononuclear cells and for methylthioinosine nucleotides in red blood cells. Measurement of the low concentrations of mono-, di- and triphosphate thioguanine nucleotides in red blood cells (detection limit 20 pmol/10⁹ cells) is possible after extraction with methanol and methylene chloride, followed by oxidation of thioguanine nucleotides with permanganate and fluorimetric detection.