Selenomethionine induces polyamine biosynthesis in regenerating rat liver tissue

Summary. Our study was undertaken to elucidate the effects of selenomethionine (SeMet) on polyamine metabolism in regenerating rat liver tissue, as useful model of rapidly growing normal tissue. We have examined the levels of spermine, spermidine and putrescine in liver tissue. At the same time we have evaluated the activities of polyamine oxidase (PAO) and diamine oxidase (DAO), the catabolic enzymes of polyamine metabolism. The obtained results suggest that polyamine levels in regenerating liver tissue, at 7^th day after two-thirds partial hepatectomy, were higher in comparison with control group. The administration of selenomethionine to hepatectomized animals during seven days, in a single daily dose of 2.5 μg/100 g body weight, increases the amount of spermine and spermidine; the level of putrescine does not change under the influence of SeMet in regenerating rat liver tissue. PAO activity is lower in regenerating hepatic tissue than in control group. Supplementation of hepatectomized animals with SeMet significantly decreases the activity of this enzyme. DAO activity was significantly higher in hepatectomized and in operated animals treated with SeMet compared to the sham-operated and control ones. The differential sensitivity observed in our model of highly proliferating normal tissue to SeMet, compared with the reported anticancer activity of this molecule is discussed.     

Growth,morphological and biochemical changes in oxa-spermine derivative-treated MCF-7 human breast cancer cells

The growth inhibitory properties of two oxa-spermine derivatives named compound 1 and compound 2, representatives of a novel type of polyamine derivatives, were studied. Dose-response growth inhibitory curves obtained after 48h drug exposure demonstrated the much higher cytotoxic activity of compound 1 towards MCF-7 human breast cancer cells. Further experiments with compound 1 showed that this oxa-spermine derivative exhibited considerable cytotoxicity with IC(50) values of 3.74 microM and 2.93 microM after 24h and 48h drug exposure respectively. In MCF-7 cells, after 8h drug (10 microM) exposure it caused shrinkage, chromatin condensation and nuclear fragmentation. However, no clear DNA laddering was detected in treated cells. Drug treatment provoked an increase in polyamine oxidase (PAO) activity. This enzyme is able to produce cytotoxic H(2)O(2) and 3-acetamidopropanal, catalyzing the oxidative deamination of N(1)-acetylated derivatives of spermine and spermidine to spermidine and putrescine respectively. Taken together these data demonstrate that the novel oxa-polyamine derivative compound 1 has considerable cytotoxic activity towards MCF-7 cells and indicate that an induction of PAO may be involved in its cytotoxic and apoptotic effects.     

Effects of glucocorticoids on polyamine metabolism in liver and spleen of guinea pig during sensitization

Summary. Glucocorticoids are potent anti-inflammatory and immunosuppressive agents. As endogenous inhibitors of cytokine synthesis, glucocorticoids suppress immune activation and uncontrolled overproduction of cytokines, preventing tissue injury. Also, polyamine spermine is endogenous inhibitor of cytokine production (inhibiting IL-1, IL-6 and TNF synthesis). The idea of our work was to examine dexamethasone effects on the metabolism of polyamines, spermine, spermidine and putrescine and polyamine oxidase activity in liver and spleen during sensitization of guinea pigs. Sensitization was done by application of bovine serum albumin with addition of complete Freund’s adjuvant. Our results indicate that polyamine amounts and polyamine oxidase activity increase during immunogenesis in liver and spleen. Dexamethasone application to sensitized and unsensitized guinea pigs causes depletion of polyamines in liver and spleen. Dexamethasone decreases polyamine oxidase activity in liver and spleen of sensitized guinea pigs, increasing at the same time PAO activity in tissues of unsensitized animals.     

Spatial organization of three-dimensional cocultures of adriamycin-sensitive and -resistant human breast cancer MCF-7 cells

Genetic and cellular heterogeneity is one of mechanisms involved in increasing tumour aggressiveness during neoplastic progression. Development of drug-resistant tumour cell subpopulations is a major problem in clinical oncology. Multi-drug resistant tumour cells survive when exposed to cytotoxic agents. Here, we studied in a three-dimensional (3D) coculture system, called "ex vivo nodules", how drug-resistant and sensitive tumour cells settle down in a 3D space. For this, we cocultured adriamycin-sensitive (MCF-7S) and -resistant (MCF-7R) human breast cancer cells in long term nodules. We showed that both types of cells are able to grow separately or in coculture until five weeks in spheroidal forms. MCF-7R cells did not loose their multi-drug resistance when cultured in nodules as measured by RT-PCR. Curiously, the exterior aspects of mixed (MCF-7S/ MCF-7R) nodules and MCF-7R nodules were similar whereas MCF-7S nodules were completely different. Nevertheless, morphologically these three nodule types were distinct, in particular in their density. Immunostaining showed that in mixed nodules, MCF-7R cells were arranged at the periphery, whereas the MCF-7S cells are in the central part of the nodules.Even if the mechanism of this arrangement remained unclear, this work shows that three-dimensional cell culture is well adapted to the study of the relationships between adhesion mechanisms and drug-resistance.     

Androgenic sensitivity of polyamine-oxidizing enzyme activities in female rat tissues

Summary. Effects of testosterone (10 μg/100 g body weight) on polyamine-oxidizing enzyme activities in female rat uterus, liver and kidney were demonstrated. Testosterone-treated rats exhibited 2.07 fold (p < 0.002) higher uterine polyamine oxidase (PAO) activity and 1.93 fold (p < 0.02) higher diamine oxidase (DAO) activity, as compared to the controls. In the liver, testosterone caused an elevation in PAO (1.39 fold, p < 0.05), but not in DAO activity, whereas in kidney the hormone stimulated DAO (1.30 fold, p < 0.05), but not PAO activity. The effects observed suggest a possible role for testosterone in the modulation of polyamine levels in the female organs studied and especially in uterus. Received May 12, 1999, Accepted December 16, 1999     

Enhancement of transglutaminase activity and polyamine depletion in B16-F10 melanoma cells by flavonoids naringenin and hesperitin correlate to reduction of the <Emphasis Type="Italic">in vivo</Emphasis> metastatic potential

Summary. The in vitro and in vivo effects of two flavonons, naringenin (NG) and hesperitin (HP) on the proliferation rate of highly metastatic murine B16-F10 melanoma cell were investigated. NG or HP treatment of melanoma cells produced a remarkable reduction of cell proliferation, paralleled with both the lowering of the intracellular levels of polyamine, spermidine and spermine and the enhancement of transglutaminase (TGase, EC 2.3.2.13) activity. Orally administered NG or HP in C57BL6/N mice inoculated with B16-F10 cells affected the pulmonary invasion of melanoma cells in an in vivo metastatic assay. The number of lung metastases detected by a computerized image analyzer was reduced, compared to untreated animals, by about 69% in NG-treated mice and by about 36% in HP-treated mice. Survival studies showed that 50% of the NG-treated animals died 38 ± 3.1 days after tumor cell injection (control group: 18 ± 1.5 days) and HP-treated mice died 27 ± 2.3 days after cell inoculation. Taken together, these findings provide further evidences for the potential anticancer properties of dietary flavonoids as chemopreventive agents against malignant melanoma.     

Sulfenic acid in human serum albumin

Summary. Sulfenic acid (RSOH) is a central intermediate in both the reversible and irreversible redox modulation by reactive species of an increasing number of proteins involved in signal transduction and enzymatic pathways. In this paper we focus on human serum albumin (HSA), the most abundant plasma protein, proposed to serve antioxidant functions in the vascular compartment. Sulfenic acid in HSA has been previously detected using different methods after oxidation of its single free thiol Cys34 through one- or two-electron mechanisms. Since recent evidence suggests that sulfenic acid in HSA is stabilized within the protein environment, this derivative represents an appropriate model to examine protein sulfenic acid biochemistry, structure and reactivity. Sulfenic acid in HSA could be involved in mixed disufide formation, supporting a role of HSA-Cys34 as an important redox regulator in extracellular compartments.     

Novel thiosemicarbazides induced apoptosis in human MCF-7 breast cancer cells via JNK signaling

Abstract

In this study, novel thiosemicarbazides and 1,3,4-oxadiazoles were synthesized and evaluated for their anticancer effects on human MCF-7 breast cancer cell lines. Among the synthesized derivatives studied, compound 2-(3-(4-chlorophenyl)-3-hydroxybutanoyl)-N-phenylhydrazinecarbothioamide 4c showed the highest cytotoxicity against MCF-7 breast cancer cells as it reduced cell viability to approximately 15% compared to approximately 25% in normal breast epithelial cells. Therefore, we focused on 4c for further investigations. Our data showed that 4c induced apoptosis in MCF-7 cells which was further confirmed by TUNEL assay. Western blotting analysis showed that compound 4c up-regulated the pro-survival proteins Bax, Bad and ERK1/2, while it down-regulated anti-apoptotic proteins Bcl-2, Akt and STAT-3. Additionally, 4c induced phosphorylation of SAPK/JNK in MCF-7 cells. Pretreatment of MCF-7 cells with 10?µM of JNK inhibitor significantly reduced 4c-induced apoptosis. Molecular docking results suggested that compound 4c showed a binding pattern close to the pattern observed in the structure of the lead fragment bound to JNK1. Collectively, the data of current study suggested that the thiosemicarbazide 4c might trigger apoptosis in human MCF-7 cells by targeting JNK signaling.     

The physiological role of biogenic amines redox reactions in mitochondria. New perspectives in cancer therapy

Summary. In tumours, polyamines and amine oxidases increase as compared to normal tissues. Cytotoxicity induced by bovine serum amine oxidase (BSAO) and spermine is attributed to H₂O₂ and aldehydes produced by the reaction. Increasing the incubation temperature from 37 to 42 °C enhances cytotoxicity in cells exposed to spermine metabolites. The combination BSAO/spermine prevents tumour growth, particularly well if the enzyme has been conjugated with a biocompatible hydrogel polymer. Since the tumour cells release endogenous substrates of BSAO, the administration of spermine is not required. Combination with hyperthermia improves the cytocidal effect of polyamines oxidation products. Our findings show that multidrug resistant (MDR) cells are more sensitive to spermine metabolites than their wild-type counterparts, due to an increased mitochondrial activity which induces the generation of intracellular ROS prior to the onset of mitochondrial permeability transition (MPT). It makes this new approach attractive, since the development of MDR is one of the major problems of conventional cancer therapy.     

IL-12重组质粒联合骨髓间充质干细胞对MCF-7人乳腺癌细胞侵袭及裸鼠移植瘤生长的影响分析

目的探讨IL-12重组质粒联合骨髓间充质干细胞（BMSC）对MCF-7人乳腺癌细胞侵袭及裸鼠移植瘤生长的影响。 方法分离纯化培养BMSC原代细胞,制备BMSC条件培养基,分析BMSC条件培养基对MCF-7细胞株体外增殖和凋亡的影响;建立裸鼠MCF-7转移瘤模型,分为对照组、BMSC组、IL-12组以及联合组,各组每3 d进行一次瘤内注射,共注射3次,15 d后处死裸鼠,比较各组裸鼠肿瘤、脾脏重量。采用χ²检验、t检验以及方差分析进行统计学分析。 结果BMSC条件培养基能够抑制MCF-7细胞增殖,且随着作用时间的延长其抑制作用升高[24 h（13.42±1.93）%,48 h（16.83±1.77）%,72 h（20.21±2.01）,F = 6.271,P = 0.000]。BMSC条件培养基处理MCF-7细胞,可有效促进MCF-7细胞凋亡,且随着作用时间的延长细胞凋亡率升高[24 h（10.82±2.18）%,48 h（18.73±2.95）%,72 h（28.15±3.52）%,F = 9.215,P = 0.000]。与对照组裸鼠肿瘤体积[1 d（124.12±9.28）mm³,3 d（582.41±17.28） mm³,7 d（983.42±42.10） mm³,15 d（793.46±109.38） mm³]比较,BMSC组[1 d（132.61±12.41） mm³,3 d（378.46±23.08） mm³,7 d（542.61±58.49）mm³,15 d（329.48±47.51） mm³]、IL-12组[1 d（119.85±13.10） mm³,3 d（322.75±26.49） mm³,7 d（518.37±67.54）mm³,15 d（302.17±68.53） mm³]以及联合组[1 d（123.41±8.94）mm³,3 d（217.85±24.03）mm³,7 d（318.29±47.32） mm³,15 d（189.27±37.58）mm³]裸鼠肿瘤体积生长速度均减慢,差异具有统计学意义（F_1d=0.827,F_3d=37.583、F_7d=31.472、F_15d=15.372,P < 0.001）;处死各组裸鼠后裸鼠肿瘤质量比较,BMSC组[（529.42±98.74） mg]、IL-12组[（544.01±117.85）mg]以及联合组[（327.55±78.56） mg]均低于对照组[（877.42±120.11）mg],且联合组裸鼠肿瘤质量最低,差异具有统计学意义（F = 10.821,P < 0.001）;而裸鼠脾指数BMSC组（7.58±1.21）、IL-12组（7.63±1.09）以及联合组（10.03±1.52）均高于对照组（5.37±0.89）,联合组裸鼠脾质量最高,差异具有统计学意义（F = 13.411,P < 0.001）。 结论BMSC在体内外均具有抑制MCF-7肿瘤增殖作用,联合使用pcDNA6/IL-12对裸鼠MCF-7转移瘤抑制具有着明显的协同作用。     

Effect of resveratrol on the radiosensitivity of 5-FU in human breast cancer MCF-7 cells

The aim of this study was to assess the efficacy of resveratrol (Res) on radiosensitivity of 5-fluorouracil (5-FU) in the spheroid culture of MCF-7 breast cancer cell line using colony formation examination. Spheroids on day 9 with 300 µm diameters were treated with 20 µM resveratrol and/or 1 µM 5-FU for one volume doubling time (VDT) (42 hours) and then irradiated with 2 Gy gamma radiation (⁶⁰Co) in various groups. Then the viability of the cells and clonogenic ability were acquired by blue dye exclusion and colony formation assay, respectively. The population doubling time in the monolayer culture and the VDT of spheroid culture was 22.48 0.23 hours and 42 0.63 hours respectively. None of the drugs and combination of them had any effect on the viability of cells. The combination treatment of 5-FU+Res+ radiation significantly reduced the colony formation ability of spheroid cells in comparison with each treatment alone. Our results indicated that resveratrol can significantly decrease colony number of breast cancer spheroid cells treated with 5-FU in combination with gamma-rays. Thus, resveratrol as a hypoxia-inducible factor-1-alpha inhibitor increased the radiosensitization of breast cancer spheroid cells.     

Induction of GPR40 positively regulates cell motile and growth activities in breast cancer MCF-7 cells

Abstract

Free fatty acid (FFA) receptors belong to a member of G-protein-coupled receptors. GPCR 120 (GPR120) and GPR40 are identified as FFA receptors and activated via the binding of long- and medium-chain FFAs. The aim of this study was to assess the effects of GPR120 and GPR40 on cell motility and growth in breast cancer cells treated with tamoxifen (TAM). MCF-7 cells were continuously treated with TAM for approximately 6?months. The expression level of GPR40 gene was markedly higher in the long-term TAM treated (MCF-TAM) cells than in MCF-7 cells. In cell motility assay, MCF-TAM cells indicated the high cell motile activity, compared with MCF-7 cells. The cell motile activity of MCF-TAM cells was suppressed by a selective GPR40 antagonist, GW1100. To evaluate the effects of GPR40 on cell growth activity under estrogen-free conditions, cells were maintained in serum-free DMEM without phenol red for 2?days. In estrogen-free conditioned medium, the cell growth rate of MCF-TAM cells was significantly higher than that of MCF-7 cells. In addition, treatment of GW1100 reduced the cell growth rate of MCF-TAM cells. These results suggest that the cell motile and growth activities may be positively regulated through the induction of GPR40 by the long-term TAM treatment in MCF-7 cells.     

New perspectives on the role of amine oxidases in physiopathology

Summary.  In the paper here presented we summarize some results obtained in our laboratory in the last few years on new structural and functional aspects of some amine oxidases (AOs), which have to be taken into consideration in defining new strategies of controlling the cellular physiopathology. In particular, the ability of Cu-AO purified from vegetal sources or from bovine serum to bind different cellular targets inducing in them conformational as well as chemical modifications are described and the consequences of this interaction on cellular functions are discussed. This is the case of the protective effect of Cu-AO against the damage induced by free radicals, cell enrichment with Cu-AO, induction of cataract and the leukocyte-endothelia interaction. The role of Cu and FAD-amine oxidases related as to the protection or damage of cells is also discussed. In this context the involvement of MAOs in the modulation of the mitochondrial functions and in the induction of apoptosis is described and some aspects of the molecular mechanism of AO inhibition by H₂O₂ and metronidazole analyzed. Received January 9, 2002 Accepted June 27, 2002 Published online November 14, 2002 Acknowledgements This paper was supported by C.N.R. grant n° G002FD1 and MURST grant (Giovani Ricercatori, 2000). Authors' address: Prof. Bruno Mondovì, Department of Biochemical Science, University of Rome “La Sapienza”, P.le Aldo Moro 5, I-00185 Rome, Italy     

Estrogen-like properties of brominated analogs of bisphenol A in the MCF-7 human breast cancer cell line

Tetrabromobisphenol A (TeBBPA) is a four-meta-brominated variant of bisphenol A (BPA) and is one of the most commonly used brominated flame retardants worldwide. We compared the estrogenic potency of TeBBPA, BPA and the brominated analogs mono- (MBBPA), di- (DBBPA), and tribromobisphenol A (TrBBPA) in the estrogen-dependent human breast cancer cell line MCF-7. All of the compounds competed with 17β-estradiol for binding to the estrogen receptor, although the affinity of the test chemicals to the estrogen receptor was much lower than that of 17β-estradiol. TrBBPA and TeBBPA showed a considerably lower access to the estrogen receptors within intact MCF-7 cells incubated in 100% serum compared to incubation in serum-free medium, indicating a strong binding to serum proteins. BPA, MBBPA, and DBBPA showed only a slightly reduced access to the receptors. All of the test compounds induced proliferation in MCF-7 cells, the potential decreasing with increasing number of bromo-substitutions. TeBBPA did not induce maximal cell growth, indicating cytotoxic effects at high concentrations. BPA and the brominated analogs, except TeBBPA, induced progesterone receptor and pS2 to the same extent as 17β-estradiol, although at much higher concentrations. Our studies demonstrate that compared to 17β-estradiol, BPA and the brominated analogs have much lower estrogenic potencies for all of the endpoints tested, TeBBPA being the least estrogenic compound. This revised version was published online in July 2006 with corrections to the Cover Date.     

抑制PI3K信号通路促进TRAIL诱导人乳腺癌细胞MCF-7凋亡

肿瘤坏死因子相关凋亡诱导配体（tumor necrosis factor-related apoptosis-inducing ligand, TRAIL）对癌细胞有独特的细胞毒性作用,而对正常细胞没有影响. 但乳腺癌细胞耐受TRAIL诱导凋亡.本研究探索磷脂酰肌醇-3激酶（phosphatidylinositol 3-kinase,PI3K）信号通路对人乳腺癌MCF-7细胞耐受TRAIL的影响. 采用MTT法、显微照相以及DAPI染色观察TRAIL对MCF-7细胞生长的抑制作用以及诱导细胞凋亡状况;流式细胞分析细胞凋亡的情况;激光共聚焦显微镜观察多聚ADP核糖多聚酶-1（poly(ADP-ribose) polymerase -1,PARP-1）的迁移和定位;Western印迹分析死亡受体、caspase-3/8、磷酸化的AKT［pAKT(Ser473)］、Src和PARP-1等蛋白质表达. 结果显示,小剂量TRAIL（< 80 nmol/L）和Ly294002（< 40μmol/L）对MCF-7细胞生长没有显著的抑制作用,但是大剂量TRAIL（160 nmol/L）和Ly294002（80 μmol/L）则能抑制MCF-7细胞生长;低剂量Ly294002协同TRAIL抑制MCF-7细胞生长,并诱导细胞凋亡;Ly294002和TRAIL共同作用能促进PARP-1从胞浆进入细胞核;蛋白质表达分析显示,MCF-7细胞均表达死亡受体DR4、DR5、诱骗受体DcR1和DcR2、以及caspase-8,但是不表达caspase-3;Ly294002和TRAIL共同作用也能抑制pAKT(Ser473)和Src的表达,并且导致PARP-1断裂. 本研究结果提示,抑制PI3K信号可增加MCF-7细胞对TRAIL诱导的敏感性;MCF-7细胞通过PI3K/AKT途径促进Src的表达耐受TRAIL的细胞毒性作用Ly294002联合TRAIL是一种新的药物组合方式治疗乳腺癌.     

Sphingosine kinase type 1 promotes estrogen-dependent tumorigenesis of breast cancer MCF-7 cells

The sphingolipid metabolite, sphingosine-1-phosphate (S1P), formed by phosphorylation of sphingosine, has been implicated in cell growth, suppression of apoptosis, and angiogenesis. In this study, we have examined the contribution of intracellular S1P to tumorigenesis of breast adenocarcinoma MCF-7 cells. Enforced expression of sphingosine kinase type 1 (SPHK1) increased S1P levels and blocked MCF-7 cell death induced by anti-cancer drugs, sphingosine, and TNF-alpha. SPHK1 also conferred a growth advantage, as determined by proliferation and growth in soft agar, which was estrogen dependent. While both ERK and Akt have been implicated in MCF-7 cell growth, SPHK1 stimulated ERK1/2 but had no effect on Akt. Surprisingly, parental growth of MCF-7 cells was only weakly stimulated by S1P or dihydro-S1P, ligands for the S1P receptors which usually mediate growth effects. When injected into mammary fat pads of ovariectomized nude mice implanted with estrogen pellets, MCF-7/SPHK1 cells formed more and larger tumors than vector transfectants with higher microvessel density in their periphery. Collectively, our results suggest that SPHK1 may play an important role in breast cancer progression by regulating tumor cell growth and survival.