Aurelio Lorico

Inhibitors of ribonucleotide reductase. Comparative effects of amino- and hydroxy-substituted pyridine-2-carboxaldehyde thiosemicarbazones.

A new series of alpha-(N)-heterocyclic carboxaldehyde thiosemicarbazones (HCTs) was studied for their effects on L1210 cell growth in culture, cell cycle transit, nucleic acid biosynthesis and ribonucleotide reductase activity. 3-Aminopyridine-2-carboxaldehyde thiosemicarbazone (3-AP) and 3-amino-4-methylpyridine-2-carboxaldehyde thiosemicarbazone (3-AMP) were the most active compounds tested with respect to inhibition of cell growth and ribonucleotide reductase activity. 5-Aminopyridine-2-carboxaldehyde thiosemicarbazone (5-AP) and 4-methyl-5-aminopyridine-2-carboxaldehyde thiosemicarbazone (5-AMP) were slightly less active. 3-AP, 3-AMP, 5-AP and 5-AMP inhibited the incorporation of [3H]thymidine into DNA without affecting the rate of incorporation of [3H]uridine into RNA. The uptake and incorporation of [14C]cytidine into cellular ribonucleotides and RNA, respectively, were not decreased by 3-AP or 3-AMP; however, the incorporation of cytidine into DNA via ribonucleotide reductase was inhibited markedly. Thus, a pronounced decrease in the formation of [14C]deoxyribonucleotides from radioactive cytidine occurred in the acid-soluble fraction of 3-AP- and 3-AMP-treated L1210 cells. Consistent with an inhibition of DNA replication that occurred at relatively low concentrations of 3-AP and 3-AMP, cells gradually accumulated in the S-phase of the cell cycle; at higher concentrations of 3-AP and 3-AMP, a more rapid accumulation of cells in the G0/G1 phase of the cell cycle occurred, with the loss of the S-phase population, implying that a second less sensitive metabolic lesion was created by the HCTs. N-Acetylation of 3-AMP resulted in a compound that was 10-fold less active as an inhibitor of ribonucleotide reductase activity and 8-fold less active as an inhibitor of L1210 cell growth. N-Acetylation of either 5-AP or 5-AMP did not alter the inhibitory properties of these compounds. The results obtained provide an experimental rationale for the further development of the HCTs, particularly 3-AP and 3-AMP, as potential drugs for clinical use in the treatment of cancer.

Apparent Lack of Mrp1-Mediated Efflux at the Luminal Side of Mouse Blood-Brain Barrier Endothelial Cells

AbstractPurpose. The purpose of this work was to determine mrp1-mediated efflux across the luminal membrane of endothelial cells at the blood-brain barrier (BBB) in mice. Methods. The transport of radiolabeled etoposide, 17β-estradiol-D-17β-glucuronide (E217βG), vincristine, and doxorubicin across the BBB of mrp1(–/–) and wild-type mice was evaluated by in situ brain perfusion. Etoposide transport was also determined in P-glycoprotein-deficient mdr1a(–/–) mice perfused with both etoposide and mrp1 inhibitors like probenecid or MK571. Cerebral vascular volume was determined by co-perfusion with labeled sucrose. Results. Sucrose perfusion indicated that the vascular space was close to normal in all the studies, indicating that the BBB remained intact. The transport of etoposide, E217βG, vincristine, and doxorubicin into the brain was not affected by the lack of mrp1. Trans-efflux studies in mrp1-deficient mice with etoposide and E217βG confirmed that mrp1 was not involved in the efflux of these substrates across the BBB. There was also a significant P-gp-mediated efflux of etoposide in studies with P-glycoprotein-deficient mdr1a(–/–) mice. Perfusion of mdr1a(–/–) mice etoposide plus probenecid or MK571 did not affect the brain transport of etoposide. Conclusion. Efflux mediated by mrp1 does not seem to occur across the luminal membrane of the endothelial cells forming the mouse BBB.

Biochemical characterisation of elsamicin and other coumarin-related antitumour agents as potent inhibitors of human topoisomerase II

Elsamicin (EM) is a recently discovered antitumour agent that is structurally related to several other compounds displaying anticancer activities, including chartreusin (CT), chrysomycin V (CV) and M (CM), gilvocarcin V (GV) and ravidomycin (RM). The biochemical events resulting in cytotoxicity for most of these compounds have not been clearly elucidated. There is some evidence that GV and CT bind to DNA and that GV is photosensitive, causing DNA damage. Therefore, we investigated the effects of these chemicals on DNA in cells and on pBR322 plasmid DNA. Using alkaline elution techniques, we found that all these compounds induced, to a different extent, DNA breakage in the human lung adenocarcinoma A549 cell line. In addition, all either bound to or intercalated into DNA, as indicated by their ability to alter the electrophoretic migration of DNA in agarose gels. Using the P4 unknotting assay, EM, CT, CV, CM, GV and RM were found to be potent inhibitors of the catalytic activity of topoisomerase II (topo II). Their potencies were compared with the known topo II inhibitors teniposide (VM-26) and doxorubicin (DX). EM was the most potent, with an IC50 of 0.4 mumol/l followed in order by CV, GV, and CT. VM-26 was the least potent with an IC50 of 15 mumol/l. It was concluded from these results that EM, GV, CV, CM and CT are capable of inhibiting topo II and that EM is the most potent inhibitor of topo II yet discovered.

Phenotypic heterogeneity of breast cancer stem cells.

Many types of tumors are organized in a hierarchy of heterogeneous cell populations, with only a small proportion of cancer stem cells (CSCs) capable of sustaining tumor formation and growth, giving rise to differentiated cells, which form the bulk of the tumor. Proof of the existence of CSC comes from clinical experience with germ-cell cancers, where the elimination of a subset of undifferentiated cells can cure patients (Horwich et al., 2006), and from the study of leukemic cells (Bonnet and Dick, 1997; Lapidot et al., 1994; and Yilmaz et al., 2006). The discovery of CSC in leukemias as well as in many solid malignancies, including breast carcinoma (Al-Hajj et al. 2003; Fang et al., 2005; Hemmati et al., 2003; Kim et al., 2005; Lawson et al., 2007; Li et al., 2007; Ricci-Vitiani et al., 2007; Singh et al., 2003; and Xin et al., 2005), has suggested a unifying CSC theory of cancer development. The reported general insensitivity of CSC to chemotherapy and radiation treatment (Bao et al., 2006) has suggested that current anticancer drugs, which inhibit bulk replicating cancer cells, may not effectively inhibit CSC. The clinical relevance of targeting CSC-associated genes is supported by several recent studies, including CD44 targeting for treatment of acute myeloid leukemia (Jin et al., 2006), CD24 targeting for treatment of colon and pancreatic cancer (Sagiv et al., 2008), and CD133 targeting for hepatocellular and gastric cancer (Smith et al., 2008). One promising approach is to target CSC survival signaling pathways, where leukemia stem cell research has already made some progress (Mikkola et al., 2010).

Biochemical and biological characterization of exosomes containing prominin-1/CD133

Exosomes can be viewed as complex “messages” packaged to survive trips to other cells in the local microenvironment and, through body fluids, to distant sites. A large body of evidence indicates a pro-metastatic role for certain types of cancer exosomes. We previously reported that prominin-1 had a pro-metastatic role in melanoma cells and that microvesicles released from metastatic melanoma cells expressed high levels of prominin-1. With the goal to explore the mechanisms that govern proteo-lipidic-microRNA sorting in cancer exosomes and their potential contribution(s) to the metastatic phenotype, we here employed prominin-1-based immunomagnetic separation in combination with filtration and ultracentrifugation to purify prominin-1-expressing exosomes (prom1-exo) from melanoma and colon carcinoma cells. Prom1-exo contained 154 proteins, including all of the 14 proteins most frequently expressed in exosomes, and multiple pro-metastatic proteins, including CD44, MAPK4K, GTP-binding proteins, ADAM10 and Annexin A2. Their lipid composition resembled that of raft microdomains, with a great enrichment in lyso-phosphatidylcholine, lyso-phosphatidyl-ethanolamine and sphingomyelin. The abundance of tetraspanins and of tetraspanin-associated proteins, together with the high levels of sphingomyelin, suggests that proteolipidic assemblies, probably tetraspanin webs, might be the essential structural determinant in the release process of prominin-1 of stem and cancer stem cells. Micro-RNA profiling revealed 49 species of micro-RNA present at higher concentrations in prom1-exo than in parental cells, including 20 with cancer-related function. Extensive accumulation of prom1-exo was observed 3 h after their addition to cultures of melanoma and bone marrow-derived stromal cells (MSC). Short-term co-culture of melanoma cells and MSC resulted in heterologous prominin-1 transfer. Exposure of MSC to prom1-exo increased their invasiveness. Our study supports the concept that specific populations of cancer exosomes contain multiple determinants of the metastatic potential of the cells from which they are derived.

Overexpression of the multidrug resistance genes mdr1, mdr3, and mrp in L1210 leukemia cells resistant to inhibitors of ribonucleotide reductase

L1210 MQ-580 is a murine leukemia cell line resistant to the cytotoxic activity of the alpha-(N)-heterocyclic carboxaldehyde thiosemicarbazone class of inhibitors of ribonucleotide reductase. The line is cross-resistant to etoposide, daunomycin, and vinblastine. L1210 MQ-580 cells expressed 8-fold resistance to 3-aminopyridine-2-carboxaldehyde thiosemicarbazone (3-AP), a relatively newly developed inhibitor of ribonucleotide reductase. The accumulation of [14C]3-AP by L1210 MQ-580 cells was 5- to 6-fold less than by parental L1210 cells. An increased rate of efflux of 3-AP was responsible for the lower steady-state concentration of 3-AP in resistant cells. In reverse transcription-polymerase chain reaction assays, L1210 MQ-580 cells were found to overexpress the multidrug resistance genes mdr1, mdr3, and mrp, but not the mdr2 gene, compared with parental L1210 cells. Measurement of the steady-state concentration of doxorubicin, a potential substrate for both the mdr and mrp gene products, demonstrated that L1210 MQ-580 cells accumulated 4-fold less anthracycline than parental cells. These findings indicate that drug efflux is a major determinant of the pattern of cross-resistance of L1210 MQ-580 cells. To extrapolate these observations to the human homologues of the mdr1, mdr3, and mrp murine genes, the effects of 3-AP were measured in L1210/VMDRC0.06 and NIH3T3 36-8-32 cells transfected with human MDR1 and MRP cDNAs, respectively. The transfectants were 2- to 3-fold resistant to the cytotoxic effects of 3-AP and accumulated less [14C]3-AP than their parental mock-transfected counterparts. Moreover, the cytotoxic activity of 3-AP was significantly greater in two double mrp gene knockout cell lines than in parental W 9.5 embryonic stem cells. Thus, the results suggest that 3-AP is a substrate for both the P-glycoprotein and MRP and that baseline MRP expression has the capacity to exert a protective role against the toxicity of this agent.

Structure-activity studies of novobiocin analogs as modulators of the cytotoxicity of etoposide (VP-16)

We have previously reported that the antibiotic novobiocin enhanced the toxicity of the anticancer agent etoposide (VP-16) to several drug-sensitive and -resistant tumor cell lines. The increase in VP-16 cytotoxicity produced by novobiocin was not due to the combined effects of these agents on topoisomerase II, but to inhibition by novobiocin of VP-16 efflux, which in turn led to increased accumulation of VP-16 and increased formation of potentially lethal VP-16-stabilized topoisomerase II-DNA covalent complexes. We have now identified novobiocin analogs that are essentially equivalent to novobiocin as inhibitors of the activity of topoisomerase II, but that are more potent than novobiocin (a) as modulators of the cytotoxicity of VP-16 to WEHI-3B leukemia and A549 lung carcinoma cells and (b) in increasing VP-16 accumulation in these cell lines. Thus, removal of the sugar moiety of novobiocin to form novobiocic acid enhanced the potency of the antibiotic as a modulator of VP-16, whereas the substituted coumarin ring alone (U-7587) was devoid of VP-16 modulatory activity. Modifications of the side chain of novobiocin significantly influenced modulatory activity, with cyclonovobiocic acid, which was formed from novobiocic acid by acid-catalyzed cycloaddition, being the most active in enhancing the cytotoxicity of VP-16. The increased potency of novobiocic acid and cyclonovobiocic acid as modulators of VP-16 activity was achieved with no change from novobiocin in the capacity of these analogs to inhibit the catalytic activity of mammalian topoisomerase II, indicating a change in the specificity of these analogs.

Wnt interaction and extracellular release of prominin-1/CD133 in human malignant melanoma cells

Prominin-1 (CD133) is the first identified gene of a novel class of pentaspan membrane glycoproteins. It is expressed by various epithelial and non-epithelial cells, and notably by stem and cancer stem cells. In non-cancerous cells such as neuro-epithelial and hematopoietic stem cells, prominin-1 is selectively concentrated in plasma membrane protrusions, and released into the extracellular milieu in association with small vesicles. Previously, we demonstrated that prominin-1 contributes to melanoma cells pro-metastatic properties and suggested that it may constitute a molecular target to prevent prominin-1-expressing melanomas from colonizing and growing in lymph nodes and distant organs. Here, we report that three distinct pools of prominin-1 co-exist in cultures of human FEMX-I metastatic melanoma. Morphologically, in addition to the plasma membrane localization, prominin-1 is found within the intracellular compartments, (e.g., Golgi apparatus) and in association with extracellular membrane vesicles. The latter prominin-1-positive structures appeared in three sizes (small, ≤40 nm; intermediates ~40-80 nm, and large, >80 nm). Functionally, the down-regulation of prominin-1 in FEMX-I cells resulted in a significant reduction of number of lipid droplets as observed by coherent anti-Stokes Raman scattering image analysis and Oil red O staining, and surprisingly in a decrease in the nuclear localization of beta-catenin, a surrogate marker of Wnt activation. Moreover, the T-cell factor/lymphoid enhancer factor (TCF/LEF) promoter activity was 2 to 4 times higher in parental than in prominin-1-knockdown cells. Collectively, our results point to Wnt signaling and/or release of prominin-1-containing membrane vesicles as mediators of the pro-metastatic activity of prominin-1 in FEMX-I melanoma.

Retroviral transfer of MRP1 and γ-glutamyl cysteine synthetase modulates cell sensitivity to L-buthionine-S,R-sulphoximine (BSO): New rationale for the use of BSO in cancer therapy

MRP1 (multidrug resistance protein 1) co-exports glutathione (GSH) and drug(s) and exports GSH, glucuronide, and sulphate-conjugated drugs. Human Fly-eco fibrosarcoma cells producing the MRP1-expressing retrovirus SF91MRP (Fly-eco MRP1), as well as 3T3 cells transduced with SF91MRP (3T3/MRP1), presented a decrease in intracellular GSH levels, as measured by two different methods. The enhanced export of GSH caused by the overexpression of MRP1 was partially counterbalanced by an increased rate of GSH synthesis. Fly-eco MRP1 and 3T3/MRP1 were hypersensitive to the GSH-depleting and cytotoxic activities of L-buthionine-S,R-sulphoximine (BSO), compared with their parental counterparts. In addition, the potentiation by BSO of the cytotoxic activity of chlorambucil and doxorubicin in Fly-eco MRP1 cells was greater than in parental Fly-eco cells. Although the turnover time of GSH, i.e. the theoretical time in which the entire GSH pool is resynthesised, was approximately 50% faster in Fly-eco MRP1 cells than in parental cells, this was not sufficient to fully restore the intracellular GSH level. In addition, mrp1 (-/-) mice were resistant to the GSH-depleting activity of intraperitoneally (i.p.) injected BSO, compared with mrp1 (+/+) mice. Co-transfer of the cDNAs for MRP1 and the heavy subunit of gamma-glutamyl cysteine synthetase (GCS) resulted in increased intracellular GSH levels and in high-level resistance to the GSH-depleting and cytotoxic activities of BSO. These data, and in particular the elevated single-agent cytotoxicity of BSO, provide a new rationale for the use of BSO in the treatment of MRP1-overexpressing tumours.

Structure-function relationships for a new series of pyridine-2-carboxaldehyde thiosemicarbazones on ribonucleotide reductase activity and tumor cell growth in culture and in vivo.

The synthesis of a new series of pyridine-2-carboxaldehyde thiosemicarbazones (HCTs) that have amino groups in the 3- and 5-positions has allowed the comparison of the structure/function relationships with regard to inhibition of ribonucleotide reductase activity, L1210 cell growth in culture and L1210 leukemia in vivo. 3-Aminopyridine-2-carboxaldehyde thiosemicarbazones are more active than the corresponding 3-hydroxy-derivatives. The 3-amino-2-pyridine carboxaldehyde thiosemicarbazones were also more active then the 5-amino-2-carboxaldehyde thiosemicarbazones in inhibiting ribonucleotide reductase activity and L1210 cell growth in culture and in vivo. N-Acetylation of the 3-amino derivative resulted in a compound that was much less active both in vitro and in vivo; N-acetylation of the 5-amino derivative did not alter the in vitro inhibitory properties, but did eliminate the antitumor properties in vivo. When the most active HCTs were studied in more detail, it was found that the incorporation of [3H]thymidine into DNA was inhibited completely without the inhibition of [3H]uridine incorporation into RNA. Further, the conversion of [14C]cytidine to deoxycytidine nucleotides and incorporation into DNA was inhibited by the HCTs without an effect on the incorporation of cytidine into RNA. These data support the conclusion that ribonucleotide reductase is the major site of action of these HCTs. The 3-aminopyridine-2-carboxaldehyde thiosemicarbazones emerge as strong candidates for development for clinical trials in cancer patients.