Modification by heme oxygenase inhibitor, tin protoporphyrin, of cellular differentiation of human myeloid leukemia K562 cell line.

Human myeloid leukemia K562 cells can be induced to differentiate to mature cells bidirectionally, i.e., hemin induces erythroid differentiation, while 12-O-tetradecanoylphorbol 13-acetate (TPA) induces differentiation to monocytes. TPA is also a potent inducer of heme oxygenase (HO), which catabolizes heme to biliverdin. We show here that TPA suppresses hemin-induced erythroid differentiation of K562 cells, while retinoids augment it. Further, an HO inhibitor, tin protoporphyrin (SnPP), suppresses TPA-induced K562 cell differentiation to monocytes. It was also found that co-treatment of K562 cells with SnPP and TPA induces erythroid differentiation of K562 cells, though SnPP alone or TPA alone does not induce erythroid differentiation, suggesting a role of HO in the directional switch of differentiation.     

Renal effects of 2-mercaptoacetyl-L-leucyl-L-phenylalanine, a novel selective inhibitor of neutral endopeptidase 24.11 (Neprilysin): comparison with SQ 28,603.

The effects of 2-mercaptoacetyl-L-leucyl-L-phenylalanine (MA-LF) on the activity of neutral endopeptidase and on renal hemodynamics and excretory function were investigated in experiments in vitro and in vivo. In vitro studies showed that the compound effectively inhibited purified bovine kidney neutral endopeptidase (Ki = 0.012 microM), while having slight influence on the activity of angiotensin I converting enzyme (Ki = 0.14 microM). In experiments on normal anesthetized rats (thiobutabarbital sodium salt, 100 mg/kg), IV administration of MA-LF (20 and 60 mg/kg) produced a dose-dependent increase in absolute rate and fractional excretion of sodium (+324% and +299%, respectively) and urinary flow rate (+261%), but did not change renal and systemic hemodynamics. Renal excretory effects of the new compound were comparable to those of the selective neutral endopeptidase inhibitor SQ 28,603. These results demonstrate that MA-LF is a potent neutral endopeptidase inhibitor with prominent natriuretic and diuretic properties.     

Effect of endopeptidase-24.11 inhibitors and C-ANP receptor ligand on responses evoked in arterioles of rat cremaster muscle by atrial natriuretic peptide.

1. The present study examined the effect of exogenous atrial natriuretric peptide (ANP), alone or in presence of inhibitors of the two major mechanisms for clearing ANP, metabolism by neutral endopeptidase-24.11 (NEP) and internalization by C-ANP receptors, on arteriolar responses using intravital microscopy on the rat cremaster muscle after intravenous or topical administration of the peptide. 2. Topical application of ANP (3 x 10(-10) to 3 x 10(-8) M) produced a gradual increase in arteriolar diameter. NEP inhibitors, thiorphan (30 mg kg-1, i.v.), kelatorphan (10 mg kg-1, i.v.) and retrothiorphan (25 mg kg-1, i.v.) alone, did not significantly affect vascular tone but caused significant potentiation of the arteriolar responses to topically applied ANP. 3. When given as an i.v. bolus, ANP dilates skeletal arterioles at a high dose (20 micrograms kg-1). At a lower dose (10 micrograms kg-2), ANP alone or with retrothiorphan or the C-ANP receptor ligand C-ANP (4-23) did not produce any arteriolar responses, while after the combined administration of the two inhibitors, an increase in arteriolar diameter was induced. 4. These results indicate that low doses of topically applied ANP dilate rat cremaster arterioles and that the vasodilator responses can be potentiated by NEP inhibition. When given as an i.v. bolus, a high dose of ANP can also dilate skeletal arterioles. However at a lower dose the rapid metabolism of the peptide prevents it from producing its action.     

表没食子儿茶素没食子酸酯对Reh细胞增殖及RUNX3基因甲基化状态的影响

目的:观察表没食子儿茶素没食子酸酯(EGCG)对白血病细胞系Reh细胞增殖抑制作用及对RUNX3基因启动子区甲基化状态及表达的影响,探讨Reh细胞RUNX3基因的失活机制及EGCG对RUNX3基因表达的调控。方法:体外培养Reh细胞,用5、10、15和20μg/mL EGCG与Reh细胞孵育,采用MTT法检测EGCG对Reh细胞增殖活性的影响;采用流式细胞术检测EGCG对Reh细胞凋亡的影响;采用甲基化特异性PCR(MSP)检测RUNX3基因启动子区域的甲基化情况;采用RT-PCR、Western blot法分别检测RUNX3 mRNA、蛋白的表达。结果:EGCG对Reh细胞生长有明显的抑制作用,并有时间及剂量依赖性;EGCG可促进Reh细胞发生凋亡,随着EGCG浓度的增加,细胞凋亡率逐渐增加;MSP法结果显示EGCG处理后RUNX3基因启动子高甲基化的区域发生去甲基化,EGCG能够诱导Reh细胞RUNX3基因mRNA和蛋白的重新表达,具剂量依赖性。结论:EGCG可明显抑制Reh细胞的增殖,并诱导Reh细胞中异常甲基化的RUNX3基因去甲基化,使RUNX3基因恢复表达,这可能是其抗肿瘤的重要机制。     

雷公藤甲素对人肝细胞L-02肝药酶活性的影响

目的:研究雷公藤甲素对正常人肝细胞株L-02细胞的损伤作用及其对肝CYP3A及CYP2E1蛋白表达量的影响。方法:体外培养L-02细胞,MTT法检测雷公藤甲素对L-02细胞的损伤,试剂盒测定培养基上清液中AST、ALT及AKP活性,Western-blot测定细胞中CYP3A及CYP2E1蛋白的含量。结果:雷公藤甲素对L-02细胞有损伤作用,且随雷公藤甲素浓度的增加或孵育时间的延长,其损伤作用增强,同时,培养基中AST、ALT及AKP的活性增强;雷公藤甲素诱导细胞中CYP3A蛋白表达减弱,CYP2E1蛋白表达增强。结论:雷公藤甲素体外对人正常肝细胞L-02细胞有损伤作用,且能影响肝CYP450酶系的表达。     

Control of essential hypertension with captopril, an angiotensin converting enzyme inhibitor.

1 Captopril, an orally active angiotensin converting enzyme inhibitor, was compared with hydrochlorothiazide (HCT) in the treatment of mild and moderate essential hypertension. 2 Twenty outpatients received no antihypertensive therapy for 2 weeks, after which they were given placebo for 8 weeks. Since their diastolic blood pressure remained above 100 mm Hg, they were then randomized to receive either captopril (twelve patients) or HCT (eight patients) for a 4-week titration period. If the supine diastolic blood pressure (SDBP) was normalized, (less than or equal to 90 mm Hg) by the end of titration period, the established regimen was continued for an 8-week maintenance period; if not, the alternate drug was added in increasing doses for up to 4 weeks and the combined therapy was maintained for the remaining 4 weeks. 3 After the first 4 weeks of therapy, both groups showed a statistically significant decrease in both systolic and diastolic blood pressure. Normalization of SDBP occurred in 75% of patients treated with captopril alone, and the addition of HCT produced normalization in the remainder. HCT alone resulted in normalization of SDBP in 50% of patients and the blood pressure of the remaining patients was normalized after the addition of captopril. 4 Captopril given orally, either alone or in conjunction with HCT, is an effective agent for the control of mild and moderate essential hypertension. 5 In our series the main side effects encountered were vertigo and dizziness, transient eosinophilia, a rise of BUN and or/a rise of SGPT or SGOT.     

Effect of endopeptidase-24.11 inhibition and of atrial natriuretic peptide clearance receptor ligand on the response to rat brain natriuretic peptide in the conscious rat.

1. The present studies examined the effect of (a) a specific endopeptidase-24.11 (E-24.11) inhibitor (candoxatrilat) and (b) a ligand for the atrial natriuretic peptide (ANP) clearance receptor (SC 46542) on the renal and blood pressure response to brain natriuretic peptide (BNP) in conscious rats. 2. Infusion of BNP 200 ng kg-1 min-1 for 60 min produced a small rise in urinary sodium and guanosine 3':5'-cyclic monophosphate (cyclic GMP) excretion with a non-significant fall in mean arterial blood pressure. 3. Candoxatrilat (3 mg kg-1) alone had no significant effect on sodium excretion or blood pressure but markedly potentiated the natriuretic response to BNP. 4. Similarly SC 46542 (68 micrograms kg-1; 6.8 micrograms kg-1 min-1) which produced no significant effect on its own, potentiated the natriuresis-induced by BNP, although the effect was of shorter duration compared to that of candoxatrilat. 5. The data indicate two approaches to the potentiation of the renal activity of BNP and suggest that BNP may mediate some of the activity of E-24.11 inhibitors reported in cardiac failure.     

Phloroglucinol: Antioxidant properties and effects on cellular oxidative markers in human HepG2 cell line

Phloroglucinol is an ubiquitous secondary metabolite encountered in a free state or polymerised as phlorotannins in brown macroalgae, and present in higher plants. FRAP and TEAC assays measured the antioxidant properties of phloroglucinol in non-biological conditions. Additionally, the biological effects of phloroglucinol (4–400 μM) were scrutinised using cellular oxidative stress markers, such as the generation of ROS, antioxidant defences (concentration of GSH and activities of GPx, GR and GST), and levels of MDA as a marker for lipid peroxidation. The direct effect was assessed immediately after an incubation period, whereas for the protective effect, the incubation period was followed by 3-h treatment with the pro-oxidant t-BOOH. The results indicated that despite having a higher radical scavenging capacity than Trolox after 30 min, phloroglucinol was not a suitable antioxidant standard for phlorotannins. Regarding the biological effects, phloroglucinol had no impact on cell viability, reduced levels of ROS and increased antioxidant defences in the direct treatment for most concentrations. The results of the protective effect were mitigated as phloroglucinol failed to protect from ROS generation but evoked a significant recovery of the stress-altered cellular antioxidant defences to restful conditions. Additionally, MDA levels were greatly reduced, preventing a radical chain oxidation.     

Antihypertensive and renal activity of SQ 28,603, an inhibitor of neutral endopeptidase.

SQ 28,603 is a potent and selective inhibitor of neutral endopeptidase 3.4.24.11 (NEP), an enzyme that degrades atrial natriuretic peptide (ANP). In conscious deoxycorticosterone acetate (DOCA)/salt hypertensive rats, 300 mumol/kg, i.v., of SQ 28,603 significantly lowered mean arterial pressure (MAP) from 177 +/- 12 to 154 +/- 8 mm Hg and increased urinary cyclic guanosine monophosphate (GMP) excretion from 204 +/- 70 to 1,068 +/- 326 pmol/kg/min within 2 h. Urinary sodium excretion increased within 20 min from a control 51.2 +/- 17.3 to 102.1 +/- 26.7 muEq/kg/min. Infusion of SQ 28,603 (3.7 mumol/kg/min) for 6 h in a separate group of conscious DOCA/salt hypertensive rats gradually reduced MAP from 180 +/- 7 to 142 +/- 7 mm Hg and increased urinary cyclic GMP excretion from 182 +/- 36 pmol/kg/min to 1,009 +/- 394 pmol/kg/min. Despite the continuous infusion of the inhibitor, the natriuretic response peaked during the first hour of treatment at 128 +/- 18 muEq/kg/min (vehicle = 54 +/- 10 muEq/min). Plasma ANP was significantly greater in the rats infused with SQ 28,603 than in those receiving vehicle (333 +/- 108 and 98 +/- 14 fmol/ml, respectively). SQ 28,603 also significantly reduced NEP activity by 95% in the kidneys (1.28 +/- 0.08 vs. 18.35 +/- 0.61 mumol/min after SQ 28,603 and vehicle respectively) and by 77% in the lungs (0.29 +/- 0.03 vs. 0.92 +/- 0.14 mumol/kg after SQ 28,603 and vehicle, respectively). In conclusion, inhibition of NEP activity by SQ 28,603 significantly decreased MAP and increased plasma ANP concentrations and urinary excretion of cyclic GMP in conscious DOCA/salt hypertensive rats.     

Development and properties of an etoposide-resistant human leukaemic CCRF-CEM cell line

Etoposide (VP-16) and several other unrelated anti-tumour agents appear to act by inhibiting the enzyme DNA topoisomerase II. We report here the development and characterization of an etoposide-resistant human leukaemic CCRF-CEM cell line, CEM/VP-1. The cell line was 15-fold more resistant to etoposide than the parental CEM cells and exhibited cross-resistance to other topoisomerase II inhibitors including teniposide, m-AMSA, and doxorubicin. CEM/VP-1 cells exhibited only a low level cross-resistance to the Vinca alkaloids, vinblastine and vincristine, known inhibitors of mitotic spindle formation. As a first step in defining the mechanism of resistance to etoposide, we compared the levels of topoisomerase II activity and its drug sensitivity in nuclear extracts from the resistant and sensitive CEM cells. As determined by a kinetoplast DNA decatenation assay, the level of DNA topoisomerase II activity in CEM/VP-1 nuclear extracts was approximately 2-fold lower than that in CEM cells, and the activity appeared to be resistant to inhibition by etoposide. Furthermore, the DNA topoisomerase II activity in CEM/VP-1 nuclear extracts did not promote the etoposide-dependent cleavage of pBR322 DNA observed with extract from sensitive cells. These results suggest that etoposide resistance in the CEM/VP-1 cell line may at least in part be due to an altered topoisomerase II, or associated factor, resulting in a reduced ability to induce DNA cleavage in the presence of drug.     

Comparison of a vasopeptidase inhibitor with neutral endopeptidase and angiotensin-converting enzyme inhibitors on bradykinin metabolism in the rat coronary bed

The in vitro effects of omapatrilat, a dual vasopeptidase inhibitor that simultaneously inhibits neutral endopeptidase (NEP) and angiotensin-converting enzyme (ACE), on exogenous bradykinin metabolism after a single passage through the coronary bed were compared with that of a NEP inhibitor (retrothiorphan, 25 nM), an ACE inhibitor (enalaprilat, 130 nM), and omapatrilat (25 nM). Bradykinin and inhibitors were infused into isolated Langendorff rat hearts perfused at 1 ml/min followed by reperfusion at 10 ml/min. Residual bradykinin was quantified in the coronary effluent by enzyme-linked immunosorbent assay to calculate bradykinin recovery and its kinetic parameters (Vmax/Km). Bradykinin degradation rate at 1 ml/min was 4.56 +/- 0.39 1/min per gram without inhibitors and was significantly reduced to 2.57 +/- 0.19 1/min per gram in the presence of enalaprilat, to 2.97 +/- 0.38 1/min per gram with retrothiorphan, to 1.82 +/- 0.17 1/min per gram with both enalaprilat and retrothiorphan, and to 1.14 +/- 0.35 1/min per gram with omapatrilat. In a second set of experiments, the effect of a 14-day treatment of rats with either ACE inhibitors (enalapril, quinapril, and ramipril), a NEP inhibitor (candoxatril), or omapatrilat on exogenous bradykinin metabolism was studied in Langendorff perfused hearts isolated from these long-term treated rats. In untreated rats, bradykinin degradation at a coronary perfusion of 1 ml/min was 4.35 +/- 0.41 1/min per gram. This value was reduced by 30% for the NEP inhibitor, by 50% for all ACE inhibitors, and by 75% for omapatrilat. All inhibitors administered either short term or long term significantly reduced bradykinin degradation during a single passage through the coronary bed. However, omapatrilat administration resulted in the greatest protection from bradykinin breakdown than ACE or NEP inhibitors alone.     

Establishment of a human colorectal cancer cell line P6C with stem cell properties and resistance to chemotherapeutic drugs

Aim:

Cancer stem cells have the capacity to initiate and sustain tumor growth. In this study, we established a CD44⁺ colorectal cancer stem cell line with particular emphasis on its self-renewal capacity, enhanced tumor initiation and drug resistance.

Methods:

Fresh colon cancer and paired normal colon tissues were collected from 13 patients who had not received chemotherapy or radiotherapy prior to surgery. Among the 6 single-cell derived clones, only the P6C cell line was cultured for more than 20 passages in serial culture and formed holoclones with high efficiency, and then the stemness gene expression, colony formation, tumorigenicity and drug sensitivities of the P6C cell line were examined.

Results:

Stemness proteins, including c-Myc, Oct3/4, Nanog, Lgr5, and SOX2, were highly expressed in the P6C cell line. Oct3/4-positive P6C cells mostly generated holoclones through symmetric division, while a small number of P6C cells generated meroclones through asymmetric division. P6C cells stably expressed CD44 and possessed a high capacity to form tumor spheres. A single cell-derived sphere was capable of generating xenograft tumors in nude mice. Compared to SW480 and HCT116 colorectal cancer cells, P6C cells were highly resistant to Camptothecin and 5-fluorouracil, the commonly used chemotherapeutic agents to treat colorectal cancers.

Conclusion:

We established a colorectal cancer stem cell line P6C with a high tumorigenic capacity and the characteristics of normal stem cells. It will benefit the mechanistic studies on cancer stem cells and the development of drugs that specifically target the cancer stem cells.     

Dichloroacetate-induced modulation of cellular antioxidant enzyme activities and glutathione level in the J774A.1 cells

Dichloroacetate (DCA) is used for different medical and industrial purposes and has been found to be a toxic by-product produced during the process of water chlorination. The DCA effects on superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GSH-Px) activities and glutathione (GSH) level were assessed and correlated with each other and also with cellular viabilities in J774A.1 macrophage cells. A concentration of 24 mm of DCA resulted in time-dependent decreases in cellular viability and glutathione level, and time-dependent increases in SOD activity when incubated with the cells for 24-48 h. DCA also resulted in significant increases in CAT and GSH-Px activities of the viable cells when incubated with the cells for 36 and 48 h. The changes in antioxidant enzyme activities and GSH levels were found to be strongly correlated with each other, and with cellular viabilities at different time points. While GSH did not result in any significant effects when added to the cells at concentrations ranging between 15 and 60 nmol ml(-1), it resulted in concentration-dependent increases in cellular viability when added to the DCA-treated cells, with maximal effects achieved at 45-60 nmol GSH ml(-1). However, cellular viability of the GSH + DCA treated cells remained below that of the control. Since viable cells from the DCA-treated cultures displayed significantly higher antioxidant enzyme activities compared with the control, it is concluded that those increases may have contributed to the cellular protection against DCA-induced cell death. Also, glutathione depletion has a major contribution to the observed cellular death induced by DCA.     

Monophthalates promote IL-6 and IL-8 production in the human epithelial cell line A549.

The dramatically increasing prevalence of allergic respiratory diseases may in part be due to the presence of certain immunotoxic xenobiotics in the environment. Recent studies have suggested that some plasticizers belonging to the phthalate family, and metabolites thereof, play a role in the development of allergic respiratory diseases. This is probably due to an adjuvant effect, which in some cases may be combined with an inflammatory process. The scope of the present study was to investigate the inflammatory potential of monophthalates, which are degradation products of phthalate plasticizers. The human epithelial cell line A549 was exposed to 15.6-2000 microg/ml, in two-fold dilutions, to either mono-n-butyl phthalate, monobenzyl phthalate, mono-n-octyl phthalate, mono-2-ethylhexyl phthalate, mono-iso-nonyl phthalate or mono-iso-decyl phthalate. Concentrations of the proinflammatory cytokines IL-6 and IL-8 were measured in the cell culture supernatant by ELISA.The study showed that some, but not all, monophthalates could induce a concentration-dependent increase in cytokine production, whereas, at higher concentrations, all phthalates suppressed cytokine production. Both the stimulatory and the suppressive properties were highly dependent on the length of the alkyl side chain of the monophthalate - a structure-activity relationship that is supported by recent observations in mice.     

Synergistic effect on reduction in blood pressure with coadministration of a renin inhibitor or an angiotensin-converting enzyme inhibitor with an angiotensin II receptor antagonist

The effects of coadministration of a renin inhibitor, terlakiren, and an angiotensinconverting enzyme inhibitor, captopril, with the angiotensin II receptor antagonist, losartan, on blood pressure of sodium-depleted guinea pigs were studied. Dose-response relationships for terlakiren (0.1 to 3.0 mg/kg, iv), captopril (0.03 to 1.0 mg/kg, iv), and losartan (0.1 to 6.0 mg/kg, iv) were obtained either alone or in the presence of a submaximal dose of the other inhibitor. The hypotensive response calculated for each compound individually was subtracted from the response to each dose of the combination of inhibitors, and the results showed that statistically significant synergy with terlakiren and captopril occurred in the presence of losartan. The degree of the synergy indicated that to achieve the same response on blood pressure, the dose of each drug could be decreased approximately 2- to 7-fold when administered in combination. These results indicate that by inhibiting multiple sites in the renin-angiotensin system, synergistic effects can be produced. The relative safety of each inhibitor could be improved by large reductions in dose when used concurrently. © 1994 Wiley-Liss, Inc.     

Enalapril, an inhibitor of angiotensin converting enzyme, increases the junctional conductance in isolated heart cell pairs.

The effect of enalapril and angiotensin II on junctional conductance (gj) of isolated rat heart cell pairs was investigated. It was found that enalapril (1 micrograms/ml) increases gj by 106 +/- 3.1% (SEM) (n = 20) within 4 min. The effect of enalapril on gj was not suppressed by propranolol (10(-6) M) or by a cAMP-dependent protein kinase inhibitor. Angiotensin II (1 micrograms/ml) reduced gj by 55%. These observations might indicate that an intrinsic renin-angiotensin system in heart is involved in the control of gj in cardiac muscle.     

Effects of endothelins on the human megakaryoblastic cell line MEG-01.

Some effects of endothelin-1 (ET-1) were studied on the megakaryoblastic cell line MEG-01. ET-1 induced an elevation of the intracellular levels of Ca2+([Ca2+]i) as measured with the fluorescent indicator indo-1, which consists of an initial transient increase and an ensuing sustained plateau. The plateau phase was abolished by removal of extracellular Ca2+. In addition, ET-1 induced a rapid (within 5 s) increase in the accumulation of inositol 1,4,5-trisphosphate (Ins(1,4,5)P3) and more delayed increases in Ins(1,3,4)P3 and Ins(1,3,4,5)P4, but did not modify cAMP levels. ET-1 homologues (ET-2, ET-3, sarafotoxin 6b and vasointestinal constrictor) also induced biphasic effects on [Ca2+]i. The Ca2+ elevation was concentration dependent, the order of potency being sarafotoxin 6b > ET-1 > ET-2 = vasointestinal constrictor > ET-3. The actions of ET analogs in raising [Ca2+]i were mutually exclusive, suggesting that they act through the same mechanism. These results suggest that cells of the megakaryoblast/megakaryocyte lineage are targets for endothelins.