Hypoxanthine transport in human tumour cell lines: relationship to the inhibition of hypoxanthine rescue by dipyridamole

Hypoxanthine (HPX) uptake was investigated in four human tumour cell lines previously characterised as being sensitive (ds: A549 and MCF7) or insensitive (di: COR-L23 and T-47D) to dipyridamole (DP)-induced inhibition of HPX rescue from antipurine antifolate-induced growth inhibition. The aim of the study was to determine the mechanism underlying the differential sensitivity of HPX rescue to DP. The time-course of HPX uptake in the two ds cell lines was different in comparison to the two di cell lines. The initial rate of HPX uptake in the di cell lines was more rapid than in the ds cell lines such that at 60 sec the amount of HPX taken up by the former was 2-6 times higher than that taken up by the later. The K(t) and T(max) for HPX transport in di COR-L23 cells were 870 microM and 4.75 microM/10(6) cells/min and 1390 microM and 1.78 microM/10(6) cells/min in ds A549 cells. HPX transport was not sodium-dependent in these cells. Equilibrative nucleoside transporter 2 (ENT2)-mediated thymidine transport was also higher in di cells. DP inhibited HPX uptake into ds cell lines by > or =48% and by < or =20% in the di cell lines. Competition studies with HPX and thymidine transport via ENT2 indicated an overlap between nucleoside and nucleobase transport transporters in the breast cancer cell lines (MCF7 and T-47D). These studies showed that more rapid and extensive HPX uptake, as well as reduced sensitivity to DP inhibition, is associated with the inability of DP to prevent HPX rescue from antipurine antifolate-induced growth inhibition in certain human tumour cell lines.     

Sensitivity of different cell lines to phototoxic effect of disulfonated chloroaluminium phthalocyanine.

Photodynamic therapy (PDT) is a treatment for cancer involving three key components: sensitizer, light and tissue oxygen. A sensitizer is a chemical compound that can be excited by light of a specific wavelength. Phthalocyanine ClAlPcS(2), belonging among the promising second generation of sensitizers, was evaluated as an inducer of photodamage on NIH3T3 (mouse fibroblasts), B16 (mouse melanoma), MCF7 (human breast adenocarcinoma) and G361 (human melanoma) cell lines. A semiconductor laser was used as a source for evocation of the photodynamic effect. We report the influence of various concentrations of the sensitizer in combination with laser irradiation on the photodamage of cells. Viability of cells was determined by means of molecular probes (Calcein AM and ethidium homodimer) for fluorescence microscopy. The quantitative changes of cell viability in relation to sensitizer concentrations and laser irradiation were proved by fluorometric measurement. We detected phototoxicity evoked by laser irradiated sensitizer in all studied cell lines. In addition, the viability studies showed that G361 melanoma cells and MCF7 breast adenocarcinoma cells were more sensitive than NIH3T3 mouse fibroblasts and B16 mouse melanoma to photodynamic damage induced by ClAlPcS(2).     

Mechanisms of toxicity associated with six tyrosine kinase inhibitors in human hepatocyte cell lines

Tyrosine kinase inhibitors have revolutionized the treatment of certain cancers. They are usually well tolerated, but can cause adverse reactions including liver injury. Currently, mechanisms of hepatotoxicity associated with tyrosine kinase inhibitors are only partially clarified. We therefore aimed at investigating the toxicity of regorafenib, sorafenib, ponatinib, crizotinib, dasatinib and pazopanib on HepG2 and partially on HepaRG cells. Regorafenib and sorafenib strongly inhibited oxidative metabolism (measured by the Seahorse‐XF24 analyzer) and glycolysis, decreased the mitochondrial membrane potential and induced apoptosis and/or necrosis of HepG2 cells at concentrations similar to steady‐state plasma concentrations in humans. In HepaRG cells, pretreatment with rifampicin decreased membrane toxicity (measured as adenylate kinase release) and dissipation of adenosine triphosphate stores, indicating that toxicity was associated mainly with the parent drugs. Ponatinib strongly impaired oxidative metabolism but only weakly glycolysis, and induced apoptosis of HepG2 cells at concentrations higher than steady‐state plasma concentrations in humans. Crizotinib and dasatinib did not significantly affect mitochondrial functions and inhibited glycolysis only weakly, but induced apoptosis of HepG2 cells. Pazopanib was associated with a weak increase in mitochondrial reactive oxygen species accumulation and inhibition of glycolysis without being cytotoxic. In conclusion, regorafenib and sorafenib are strong mitochondrial toxicants and inhibitors of glycolysis at clinically relevant concentrations. Ponatinib affects mitochondria and glycolysis at higher concentrations than reached in plasma (but possibly in liver), whereas crizotinib, dasatinib and pazopanib showed no relevant toxicity. Mitochondrial toxicity and inhibition of glycolysis most likely explain hepatotoxicity associated with regorafenib, sorafenib and possibly pazopanib, but not for the other compounds investigated.     

Cell cycle-dependent inhibition of the proliferation of human neural tumor cell lines by iron chelators

The current studies were designed to examine the conditions under which the ferric iron chelator desferrioxamine (DFO) arrested cell cycle progression and hence the proliferation of neural cell lines in vitro. DFO arrested proliferation at different stages of the cell cycle depending on the concentration and duration of drug exposure. Twenty-four-hour treatment with 160 μM DFO arrested glioma cells in G₁, whereas 72-hr treatment with 10 μM DFO acted to slow the passage of glioma cells through the cell cycle, eventually accumulating in G₂/M. Another iron chelator, ADR 529, also inhibited the proliferation of glioma cells by lengthening the period of the cycle and causing the cells to arrest in G₂/M. The effects of 10 and 160 μM DFO were irreversible after 24 and 48 hr, respectively, and 10 μM DFO became cytotoxic after 3 days. These observations demonstrate that DFO has different effects on the proliferation of neural tumor cell lines depending on the concentration and time of exposure, which result in different sites of cell cycle arrest. These different in vitro actions of DFO may have ramifications for the successful application of iron chelator therapy in vivo.     

Potent and selective Aurora inhibitors identified by the expansion of a novel scaffold for protein kinase inhibition

Potent and selective Aurora kinase inhibitors were identified from the combinatorial expansion of the 1,4,5,6-tetrahydropyrrolo[3,4-c]pyrazole bi-cycle, a novel and versatile scaffold designed to target the ATP pocket of protein kinases. The most potent compound reported in this study had an IC(50) of 0.027 microM in the enzymatic assay for Aur-A inhibition and IC(50)s between 0.05 microM and 0.5 microM for the inhibition of proliferation of different tumor cell lines.     

The inhibition of cell proliferation using silencing of N-cadherin gene by siRNA process in human melanoma cell lines

Malignant melanoma is a disease with high mortality rate caused by rapid metastasis. Cell motility is physically and biochemically restricted by cadherin-mediated cell interactions and signalling pathways, and alterations in cadherin expression strongly correlate with E to N-cadherin switch as well as the metastasis and progression of tumours. Contrary to E-cadherin, N-cadherin plays an important role in stimulating processes of cell division, migration, differentiation and death. In this study we investigated the role of N-cadherin in proliferation and AKT, ERK, beta-catenin signalling pathway in human melanoma cells: WM793(VGP), WM115(VGP) from the primary tumor site, as well as Lu1205(lung) and WM266-4(skin) from metastatic sites. N-cadherin, pAKT(S473), β-catenin, pERK1/2(T202/Y204), cyclin D1, cyclin D3, cyclin-dependent kinases CDK4, CDK6, and p15, p16, p21, p27 inhibitors expression was determined by western blot analysis. The study on proliferation of cells was performed with the use of BrdU incorporation and crystal violet staining assays. Knock-out of N-cadherin gene expression by siRNA process reduced the expression of: pAKT(S473), pERK1/2(T202/Y204), betacatenin, cyclin D1, cyclin D3, cyclin-dependent kinases CDK4, CDK6 while increasing expression of cell cycle inhibitors p21 and p27, and significantly decreased cell proliferation (50-70%). The collected data indicate that N-cadherin mediates the effect of cell cycle in G1 phase by AKT, β-catenin, and ERK signalling pathway. These results suggest that increased expression of N-cadherin significantly contributes to the increased invasive potential of melanoma cells. Silencing of N-cadherin arrests cell growth at G1 phase and inhibits the entry into S-phase which is of great importance as to its possible future use in cancer treatment.     

Therapeutic potential of Aurora kinase inhibitors in cancer

Aurora kinases (AKs) represent a family of serine/threonine protein kinases that regulate mitotic processes during cell division. They are primarily involved in regulating the multiple steps of mitosis, including centrosome duplication, formation of bipolar mitotic spindle, chromosome alignment on the mitotic spindle, establishment and maintenance of the spindle checkpoint and cytokinesis. As AKs are key regulators of mitosis, several studies have indicated that they have a strong association with cancer and are overexpressed in numerous cancerous cell lines as well as human malignancies. Thus, AKs represent a promising therapeutic target for anticancer drug development. In this review, the role of AKs in cancer, and the current status and therapeutic potential of AK inhibitors is discussed.     

1,4,5,6-tetrahydropyrrolo[3,4-c]pyrazoles: identification of a potent Aurora kinase inhibitor with a favorable antitumor kinase inhibition profile

The optimization of a series of 5-phenylacetyl 1,4,5,6-tetrahydropyrrolo[3,4-c]pyrazole derivatives toward the inhibition of Aurora kinases led to the identification of compound 9d. This is a potent inhibitor of Aurora kinases that also shows low nanomolar potency against additional anticancer kinase targets. Based on its high antiproliferative activity on different cancer cell lines, favorable chemico-physical and pharmacokinetic properties, and high efficacy in in vivo tumor models, compound 9d was ultimately selected for further development.     

Dawn of Aurora kinase inhibitors as anticancer drugs

With the current standard chemotherapy regimens only approximately 25% of acute myelogenous leukaemia (AML) patients survive > 5 years. Aurora kinases are overexpressed in many human cancers. VX-680 inhibited Aurora-A, -B, -C and the FMS-like tyrosine kinase-3 with apparent inhibitory constants of 0.6, 18, 4.6 and 30 nM, respectively. In primary leukaemia cells from patients with AML, which were refractory to standard therapies, VX-680 inhibited colony formation. In nude mice, VX-680 markedly reduced human AML tumours. The development of VX-680 for use in AML should continue.     

Dawn of Aurora kinase inhibitors as anticancer drugs

With the current standard chemotherapy regimens only ～ 25% of acute myelogenous leukaemia (AML) patients survive > 5 years. Aurora kinases are overexpressed in many human cancers. VX-680 inhibited Aurora-A, -B, -C and the FMS-like tyrosine kinase-3 with apparent inhibitory constants of 0.6, 18, 4.6 and 30 nM, respectively. In primary leukaemia cells from patients with AML, which were refractory to standard therapies, VX-680 inhibited colony formation. In nude mice, VX-680 markedly reduced human AML tumours. The development of VX-680 for use in AML should continue.     

不同株人肝癌细胞对化疗药物杀伤作用的敏感性分析

目的研究化疗药物对不同株人肝癌细胞杀伤作用的敏感性。方法采用MTT比色分析法测定多柔比星(DXRB)或米托蒽醌(DHAQ)对体外培养的4株人肝癌细胞的细胞毒作用。结果DXRB对SMMC-7721细胞的杀伤作用最强,QJY次之,HEPG-2和Alexander不敏感;DHAQ对QJY细胞的杀伤作用最强,Alexander和SMMC-7721次之,同样HEPG-2不敏感。结论不同株人肝癌细胞对同一化疗药物可具有不同的敏感性。     

Poly(ADPR)polymerase inhibition and apoptosis induction in cDDP-treated human carcinoma cell lines

Poly(ADPR)polymerases' (PARPs) inhibitors potentiate the cytotoxic effects of chemotherapeutic agents like alkylating compounds and TOPO I poisons, while their action in combination with cisplatin still needs investigation. In fact, one of the earliest responses to DNA single- or double-strand breaks is the synthesis of poly(ADP-ribose) (PAR) by PARPs; these enzymes are components of DNA repair machineries and substrates of caspases. Cisplatin (cDDP) yields intra- and inter-strand DNA cross-links and several proteins that recognise cDDP-induced DNA damage, such as p53, are also targets of poly(ADP-ribosyl)ation. We compared the effects of treatments with cDDP and the PARPs inhibitor PJ34 in p53 mutated carcinoma cell lines (HeLa, KB, HT29) that exhibited differential sensitivities to the drugs, in terms of cell growth inhibition and onset of apoptosis. In cDDP-resistant HT29 cells we determined: (i) PJ34 potentiation of cDDP-induced cell growth inhibition; (ii) an increment of PARP-1 automodification following cDDP treatment. In cDDP-sensitive HeLa cells, we found that the drug induced apoptotic cell death associated with caspase-dependent PARP-1 proteolysis.     

Amine uptake inhibition by diosmin and diosmetin in human neuronal and neuroendocrine cell lines.

Human neuroblastoma cells of sympathetic origin have been used for studying the effects of diosmin and its metabolite diosmetin (vasotonic agent) on amine reuptake systems. Neuroblastoma cells take up 3H-dopamine in a specific and time-dependent manner. 3H-dopamine uptake was dose-dependently inhibited by the known antagonist desipramine. Diosmin did not affect 3H-dopamine uptake at concentrations as high as 1 mM. On the other hand the aglycone metabolite of diosmin, diosmetin, inhibited 3H-dopamine uptake in a dose-dependent manner (IC50 = 4 microM). Diosmetin inhibited 3H-dopamine uptake in control and differentiated neuroblastoma cells, as well as in small-cell lung carcinoma cells. Furthermore diosmetin also inhibited 3H-serotonin uptake in both cell types. These results demonstrate that some flavonoids act as antagonists of plasma membrane amine transporters at the molecular level and suggest that inhibition of amine reuptake at the level of peripheral sympathetic nerve terminals could be responsible for the increased vascular tone observed in vivo after treatment with these drugs.     

3-methylindole-induced toxicity to human bronchial epithelial cell lines.

Transfected BEAS-2B cells that express different cytochrome P450 enzymes were used to assess whether human bronchial epithelial cell lines are target cells for 3-methylindole (3MI)-induced damage. Four different transfected BEAS-2B lines overexpressing P450s 2A6, 3A4, 2F1, and 2E1 (B-CMV2A6, B-CMV3A4, B-CMV2F1, and B-CMV2E1), respectively, were compared. The B-CMV2F1 and B-CMV3A4 cells were the most susceptible to 3MI-mediated cytotoxicity, measured by leakage of lactate dehydrogenase into the medium after a 48-h incubation. The toxicity was ameliorated by pretreatment with 1-aminobenzotriazole (ABT). Depletion of glutathione with diethylmaleate decreased the onset and increased the extent of cell death with 3MI. Thus, 3MI is cytotoxic to immortalized bronchial epithelial cells overexpressing 2F1 without concomitant depletion of GSH, but depletion of GSH modestly enhances the cytotoxicity of 3MI to human lung cells. Additional studies clearly demonstrated that a low concentration of 3MI (10 micro M) induced apoptosis in BEAS-2B cells that was measured by DNA fragmentation, and apoptosis was inhibited by the presence of ABT. The B-CMV2F1 cells overexpressing 2F1 demonstrated increased apoptosis (measured by Annexin-V binding) at 24 h with 100 micro M 3MI. Therefore, CYP2F1 in human bronchial epithelial lung cells may bioactivate 3MI to 3-methyleneindolenine, which induces programmed cell death at relatively low concentrations. Human lung cells may be susceptible to this prototypical pneumotoxicant.     

Perifosine selectively induces cell cycle block and modulates retinoblastoma and E2F1 protein levels in p53 mutated leukemic cell lines

The effect of the single-chain alkylphospholipid perifosine was analyzed in p53^wild-type (SKW6.4, OCI and MOLM), p53^mutated (BJAB, MAVER) and p53^null (HL-60) leukemic cell lines. Perifosine promoted cytotoxicity with a combination of apoptosis induction in all cell lines and cell cycle block at the G₂M checkpoint, which was selectively observed in p53^mutated BJAB and MAVER cell lines. At the molecular level, perifosine induced hypophosphorylation of retinoblastoma protein and the degradation of E2F1 protein in p53^mutated but not in p53^wild-type cells. These data indicate that perifosine potentially represents an innovative therapeutic approach for p53^mutated hematological malignancies.     

IkappaB kinase activation is involved in regulation of paclitaxel-induced apoptosis in human tumor cell lines.

Paclitaxel (Taxol), a naturally occurring antimitotic agent, has shown significant cell-killing activity against human solid tumor cells through induction of apoptosis. The molecular mechanism underlying paclitaxel-induced apoptosis is not entirely clear. Using the unique inhibitory effect of glucocorticoids on paclitaxel-induced apoptosis, we recently discovered that paclitaxel-induced inhibitor kappaBalpha (IkappaBalpha) degradation and nuclear factor-kappaB (NF-kappaB) activation might contribute to the mediation of paclitaxel-induced apoptosis. In this study, using a novel IkappaBalpha phosphorylation inhibitor, we demonstrated that the blockage of paclitaxel-induced IkappaBalpha degradation inhibited apoptotic cell death in human breast cancer BCap37 and ovarian cancer OV2008 cell lines. Furthermore, in vitro kinase assays showed that the activity of IkappaB kinase (IKK), which is responsible for the phosphorylation and degradation of IkappaB proteins, was significantly activated by paclitaxel in these paclitaxel-sensitive tumor cells. Stable transfection of a mutant IkappaBalpha lacking Ser(32) and Ser(36) that was insensitive to IKK-mediated phosphorylation and degradation resulted in reduced sensitivity of tumor cells to paclitaxel-induced apoptosis. Moreover, we also found that the expression of mitogen-activated protein kinase/extracellular signal-regulated kinase kinase kinase 1, an upstream regulator of IKK, was up-regulated by paclitaxel. These findings suggest that the activation of IKK might play a critical role in the regulation of paclitaxel-induced NF-kappaB activation that subsequently mediates paclitaxel-induced apoptotic cell death in solid tumor cells.     

Heparin inhibits dengue-2 virus infection of five human liver cell lines

Liver is suggested to be the major target of dengue virus infection and plays an important role in the immunopathogenesis of dengue hemorrhagic fever. Previously, we reported that five human liver cell lines (HuH-7, HA22T, Hep3B, PLC, and Chang liver) with various degrees of differentiation and tumorigenicity showed different susceptibility for dengue virus infection. Here, we demonstrate that heparin, an analogue of heparan sulfate (HS), can compete with HS on cell membrane for virus binding and subsequently inhibits the replication of dengue-2 and Japanese encephalitis viruses in hepatoma and BHK-21 cells, respectively. It indicates that the binding of these viruses with HS is an important process for their invasion. Moreover, the inhibitory effect of heparin correlates with the infectivity of the virus in the cells. All together, our results suggest that HS is an important host component for dengue and Japanese encephalitis virus replication, which can be effectively blocked by heparin.     

Small interfering RNA-mediated suppression of dUTPase sensitizes cancer cell lines to thymidylate synthase inhibition

Uracil misincorporation into DNA and its associated misrepair have been implicated as contributing components of cytotoxicity resulting from treatment with thymidylate synthase inhibitors. dUTPase, which eliminates dUTP from the DNA biosynthetic pathway, opposes uracil misincorporation; therefore, elevation of this enzyme in cancer cells may contribute to drug resistance. To validate the potential of dUTPase as a target for drug development, we used small interfering RNA directed against this enzyme and determined the effects of decreasing levels of dUTPase on sensitivity to the thymidylate synthase (TS) inhibitor fluorodeoxyuridine (FUdR) in human cancer cell lines. Suppression of dUTPase in SW620 and MCF-7 cells resulted in a significant enhancement in dUTP pool expansion after TS inhibition. This shift in nucleotide pool levels was accompanied by a significant decrease in the FUdR IC(50)(72h) ( approximately 75-fold for SW620 cells and approximately 6-fold for MCF-7 cells), a decline in clonogenic survival, and enhanced DNA double strand break formation. In contrast, depletion of dUTPase in HT29 cells did not substantially affect chemosensitivity or the amount of DNA damage incurred despite a 3-fold increase in dUTP pool expansion. This observation implies that the cytotoxic impact of uracil misincorporation may reach a saturation point in HT29 cells and that a further increase in dUTP levels has no additive effect. Together, these results suggest that uracil misincorporation is a potent determinant of cytotoxicity to TS inhibition and indicate that partial inhibition of dUTPase is a viable therapeutic approach to enhance the efficacy of broadly used chemotherapeutic agents that inhibit TS.     

熊去氧胆酸选择性诱导人肝肿瘤细胞凋亡及抑制增殖的实验研究

目的: 探讨熊去氧胆酸 (UDCA)对肝肿瘤细胞株诱导凋亡及抑制增殖的作用和机制。方法:用四氮唑蓝法、流式细胞术、TUNEL法、Wright Giemsa染色法、电镜及免疫细胞化学等方法,观察UDCA对肝肿瘤细胞株HepG2,BEL7402和正常人肝细胞株L 02的生长活力、细胞凋亡、细胞周期及Bax/bcl 2表达的影响。结果: UDCA对HepG2,BEL7402细胞株生长的抑制作用随药物浓度增高而增强(r2 =0. 96, P<0. 01;r2 =0. 97, P<0. 01;48h)。UDCA对HepG2及BEL7402的IC50分别为0. 92 mmol·L-1, 0. 86 mmol·L-1。UDCA(1. 0mmol·L-1 )对HepG2及BEL7402的凋亡率分别为(42±6) %及 (44±4) %,明显高于对照组(P<0. 01),并且阻滞细胞周期于S期。以UDCA(0. 8mmol·L-1 )处理HepG2 后bcl 2 表达由(24. 3±2. 4) %降低为 (10. 1±1. 6 ) %,Bax表达由(43±5) %升高为 (59±3) % (P<0. 01 );处理BEL7402细胞后bcl 2表达由 (21. 6±1. 8) %降为(11. 6±2. 1) %,Bax表达由 ( 44±4 ) %升高为(59±3) % (P<0. 01)。UDCA对L 02细胞无明显作用。结论: UDCA对HepG2, BEL7402细胞株有显著的抑制增殖及诱导凋亡作用,它可能与UDCA阻滞细胞周期、降低bcl 2和提升Bax的表达有关。