Regulation of Polyamine Synthesis and Transport by Fibroblast Growth Factor in Aortic Smooth Muscle Cells

Abstract

Basic-FGF (FGF2) is implicated as a regulator of smooth muscle cell proliferation that develops after arterial injury. Polyamines are essential for cell growth and differentiation and may mediate some of the FGF2-elicited responses. To examine this possibility, the effect of FGF2 on polyamine synthesis and uptake was tested on rat arterial smooth muscle cells. Exposure of cells to FGF2 for 24 and 48 h resulted in increased intracellular polyamine content. Ornithine decarboxylase (ODC) activity increased in FGF2-treated cells after 6 h of treatment, whereas no increases were detected in ODC mRNA steady-state levels. Basic-FGF increased maximal polyamine transport rate without changes in Km. Treatment with actinomycin D decreased polyamine transport. The effect of cyclohexamide on polyamine uptake was dose dependent. These studies indicate that treatment of vascular smooth muscle cells with FGF2 results in increases in intracellular polyamine content, polyamine synthetic activity, and polyamine transport.     

LKM512 yogurt consumption improves the intestinal environment and induces the T-helper type 1 cytokine in adult patients with intractable atopic dermatitis

BACKGROUND: In atopic dermatitis (AD) patients, the intestinal mucosal barrier function is weakened, permiting frequent invasion by antigens. Polyamines and short-chain fatty acids (SCFA) produced by intestinal bacteria are involved in the promotion of intestinal mucosal barrier functions. AIM: Our aim was to investigate the effect of pro-biotic yogurt containing Bifidobacterium animalis subsp. lactis LKM512 (LKM512 yogurt) on subjective symptoms, intestinal microbiota, intestinal bacterial metabolites (polyamines and SCFA), and T-helper type 1 (Th1)/Th2 balance in intractable AD patients. METHODS: In a double-blind, placebo-controlled, crossover study, LKM512 yogurt was given for 4 weeks to 10 adult AD patients who were diagnosed with moderate AD (four males and six females; average age, 22.1 years). The subjective symptoms were recorded after each intervention. The dynamics of fecal microbiota were analysed by the terminal-restriction fragment length polymorphism method. The effects of LKM512 yogurt on fecal polyamines, SCFA, and serum cytokines were evaluated. RESULTS: Scores of itch and burning tended to improve to a greater extent by LKM512 yogurt consumption than by placebo consumption. LKM512 yogurt (P<0.005) and placebo consumption (P<0.05) significantly increased the serum IFN-gamma concentration by six- and threefold, respectively. Fecal microbiota was altered dynamically by LKM512 yogurt consumption, in particular, the bacterial species and phylotypes of Bifidobacterium, Clostridium cluster IV and subcluster XIVa were increased in number. In addition, fecal spermidine concentration was significantly (P<0.05) increased, while fecal butyrate also tended to be increased by LKM512 yogurt consumption. CONCLUSION: We conclude that LKM512 yogurt consumption may be effective against intractable adult-type AD and this effect depends on the recovery of the intestinal mucosal barrier function and the induction of the Th1-type cytokine by polyamines and SCFA, particularly, butyrate, produced by the altered intestinal microbiota.     

Analysis of structural and biochemical events occurring in the small intestine after dietary polyamine ingestion in suckling rats

In the present investigation, we analyzed the mechanism involved in spermine-induced intestinal maturation in suckling rats. Spermine was given orally to suckling pups and biochemical as well as morphological parameters were studied at different times after the beginning of the treatment. Eight hours after administration, spermine produced cell elimination at the villus tops and a decrease in intestinal DNA and protein content. In parallel, protein and DNA concentration and disaccharidase activity were enhanced in the chyme. These transitory alterations were not induced by growth inhibition, as DNA synthesis was not modified, although a brief decrease in protein synthesis was observed. Spermine was not metabolized in cytotoxic products: rat pretreatment with MDL72527 (an inhibitor of polyamine oxidase) did not avoid the decrease in disaccharidase activity and in DNA and protein content. Three days after treatment, sucrase and maltase activity was higher in rats treated with spermine and MDL72527 than that in animals receiving spermine alone. Lactulose or acetylspermine ingestion induced intestinal maturation. Our data suggest that dietary polyamines exert a direct and specific maturational effect on rat small intestine and that an early decrease in lactase activity plays an important role in this phenomenon.     

Cytotoxicity and cell death mechanisms induced by the polyamine-vectorized anti-cancer drug F14512 targeting topoisomerase II

The polyamines transport system (PTS) is usually enhanced in cancer cells and can be exploited to deliver anticancer drugs. The spermine-conjugated epipodophyllotoxin derivative F14512 is a topoisomerase II poison that exploits the PTS to target preferentially tumor cells. F14512 has been characterized as a potent anticancer drug candidate and is currently in phase 1 clinical trials. Here we have analyzed the mechanisms of cell death induced by F14512, compared to the parent drug etoposide lacking the polyamine tail. F14512 proved to be >30-fold more cytotoxic than etoposide against A549 non-small cell lung cancer cells and triggers less but unrecoverable DNA damages. The cytotoxic action of F14512 is extremely rapid (within 3 h) and does not lead to a marked accumulation in the S-phase of the cell cycle, unlike etoposide. Interestingly, A549 cells treated with F14512 were less prone to undergo apoptosis (neither caspases-dependent nor caspases-independent pathways) or autophagy but preferentially entered into senescence. Drug-induced senescence was characterized qualitatively and quantitatively by an increased β-galactosidase activity, both by cytochemical staining and by flow cytometry. A morphological analysis by electron microscopy revealed the presence of numerous multi-lamellar and vesicular bodies and large electron-lucent (methuosis-like) vacuoles in F14512-treated cell samples. The mechanism of drug-induced cell death is thus distinct for F14512 compared to etoposide, and this difference may account for their distinct pharmacological profiles and the markedly superior activity of F14512 in vivo. This study suggests that senescence markers should be considered as potential pharmacodynamic biomarkers of F14512 antitumor activity.     

Onchocerca volvulus: immunohistochemical and immunoelectron microscopical distribution of a polyamine oxidizing enzyme

We studied the distribution of a polyamine oxidizing enzyme (PAO) in Onchocerca volvulus and other nematode parasites by immunohistochemistry and electron microscopy with immunogold technique using a polyclonal antiserum raised against purified PAO from Ascaris suum . In adult O. volvulus the protein was localized in the outer zone and the area of the basal labyrinth of the hypodermis and occasionally in the outer zone of the uterine epithelium. Further, the fluid in the body cavity was strongly stained. No specific labelling was observed in the cuticle, muscles, epithelia of intestine, ovaries, testis and vas deferens or in sperm, oocytes and embryos. Third‐stage larvae of O. volvulus in Simulium soubrense showed strong staining; the same was observed in Anisakis sp. larvae, where the inner and outer zone of the hypodermis were strongly labelled. All mature, intact and dead microfilariae in nodules, skin and lymph nodes were well stained and it was possible to show that the cytoplasm of the hypodermal cells, but not the mitochondria, nuclei or other organelles of muscle cells, was preferentially labelled by immunogold particles. Investigation of adult A. suum presented specific labelling of the hypodermis, but the basal labyrinth was more strongly marked than the outer zone.     

伯氏疟原虫感染红细胞中的游离氨基酸和多胺生成的研究

伯氏疟原虫氯喹敏感株(CS)和抗氯喹株(CR)感染红细胞中的游离氨基酸量和鸟氨酸脱羧酶活力均相接近,用氯喹10mg/kg im两个株的感染小鼠,20h后对游离氨基酸无抑制作用.氯喹剂量为5mg/kg时,抑制CS和CR感染红细胞的鸟氨酸脱羧酶活力为79.6和55.7％。 CS和CR感染红细胞中的精脒量为139±27和528±140nmol/10~9感染红细胞。环亮氨酸剂量为80mg/kg时,能抑制CS和CR感染红细胞的精脒生成,其抑制率分别为44和57％,加喂甲硫氨酸(100mg/kg)后,精脒量分别上升至294和657nmol/10~9感染红细胞。但是与两者仍有一定差距,故认为在S-腺苷甲硫氨酸合成酶及其后的代谢环节仍有差异.     

Targeting polyamine metabolism: a viable therapeutic/preventative solution for cancer?

The polyamine pathway has been identified as a target for the design of new antiproliferative drugs, due to the strong positive relationship between intracellular polyamine content and cell, particularly cancer cell growth. A number of single enzyme inhibitors have been synthesised against the two key biosynthetic enzymes, ornithine decarboxylase and S-adenosylmethionine decarboxylase, but their success in the clinic has been limited due to incomplete polyamine depletion and induction of compensatory mechanisms that counteract the effects of enzyme inhibition. Overall, clinical trials of these agents as chemotherapeutic drugs have proved disappointing, with either little clinical efficacy or unacceptable toxicity. The polyamine analogues provide an alternative strategy that shows promise, particularly against diseases other than cancer. Combination of the polyamine inhibitors with classic cytotoxic agents may be an alternative strategy that is showing some promise, at least in vitro. An avenue that is, however, presently more promising is the use of polyamine inhibitors or analogues as chemopreventative agents against a range of human cancers. It seems likely that the future use of these drugs will be in disease prevention rather than treatment. With regard to the newer agents with restricted conformation that are now undergoing clinical trials, it is too early to say whether they will be chemotherapeutic and/or chemopreventative. This article focuses on the clinical use and responses to inhibitors of polyamine metabolism.     

二氟甲基鸟氨酸对人肺癌细胞L78细胞生长抑制、凋亡诱导及相关基因的表达调控

目的：研究二氟甲基鸟氨酸(DFMO)对人肺癌细胞L78生长及相关基因表达的影响。方法：应用细胞生长曲线、端粒酶活性、细胞形态及DNA电泳观察细胞生长速率及细胞凋亡情况，同时应用Northern blot及免疫组化技术检测相关基因的表达。结果：(1)DFMO处理的L78细胞与对照组相比生长受到抑制，同时，诱导细胞凋亡增多。(2)在DFMO作用下，L78细胞df4基因被诱导表达，同时伴有P21^ras表达下调，Fas基因表达的上调。结论：DFMO抑制L78细胞生长，诱导凋亡的作用与其对df4基因的诱导密切相关，DFMO通过对df4的诱导．调控P21^ras和Fas的表达．     

Polyamine dendrimer-based MRI contrast agents for functional kidney imaging to diagnose acute renal failure

PURPOSE: To choose an efficacious renal functional MRI contrast agent to image early renal tubular damage. We synthesized and compared smaller polyamine dendrimer-based MRI contrast agents (<60 kD) that, unlike Gd-[DTPA], transiently accumulate in renal tubules and can be used to visualize renal structural and functional damage. MATERIALS AND METHODS: Six dendrimer-based MRI contrast agents smaller than 60 kD were studied by high resolution dynamic micro-MRI and compared to Gd-[DTPA]-dimeglumine and Gadomer-17. The best agent, DAB-G2, was further tested in a mouse ischemia/reperfusion model to validate its efficacy. RESULTS: Despite unequal renal clearance rates, all polyamine dendrimer agents visualized the renal functional anatomy of the mice better than Gd-[DTPA]-dimeglumine and Gadomer-17. DAB-G2 was excreted most rapidly, yet was able to visualize mild renal tubular injury very early after injury. CONCLUSION: DAB-G2 was found to be the best candidate for functional kidney imaging and enabled early diagnosis of acute renal injury.     

Enzymatic oxidation products of spermine induce greater cytotoxic effects on human multidrug-resistant colon carcinoma cells (LoVo) than on their wild-type counterparts

The occurrence of resistance to cytotoxic agents in tumor cells, associated with several phenotypic alterations, is one of the major obstacles to successful anticancer chemotherapy. A new strategy to overcome MDR of human cancer cells was studied, using BSAO, which generates cytotoxic products from spermine, H(2)O(2) and aldehyde(s). The involvement of these products in causing cytotoxicity was investigated in both drug-sensitive (LoVo WT) and drug-resistant (LoVo DX) colon adenocarcinoma cells. Evaluation of clonogenic cell survival showed that LoVo DX cells are more sensitive than LoVo WT cells. Fluorometric assay and treatments performed in the presence of catalase demonstrated that the cytotoxicity was due mainly to the presence of H(2)O(2). Cytotoxicity was eliminated in the presence of both catalase and ALDH. Transmission electron microscopic observations showed more pronounced mitochondrial modifications in drug-resistant than in drug-sensitive cells. Mitochondrial functionality studies performed by flow cytometry after JC-1 labeling revealed basal hyperpolarization of the mitochondrial membrane in LoVo DX cells. After treatment with BSAO and spermine, earlier and higher mitochondrial membrane depolarization was found in LoVo DX cells than in drug-sensitive cells. In addition, higher basal ROS production in LoVo DX cells than in drug-sensitive cells was detected by flow-cytometric analysis, suggesting increased mitochondrial activity in drug-resistant cells. Our results support the hypothesis that mitochondrial functionality affects the sensitivity of cells to the cytotoxic enzymatic oxidation products of spermine, which might be promising anticancer agents, mainly against drug-resistant tumor cells.