Cytotoxic effects of FK506 on human renal proximal tubule cells in culture

Summary FK506 has been used as the primary immunosuppressive agent administered after a variety of organ transplants, with less reported nephrotoxicity than that of cyclosporine. This study examined in vitro cytotoxicity of FK506 on normal human renal proximal tubule cells. Cytotoxicity was assessed by neutral red inclusion and trypan blue exclusion; morphology was assessed by light and transmission electron microscopy. Neutral red inclusion decreased to less than 10% of the control after 3 days exposure to 200μg/ml FK506. Forty microgram per milliliter FK506 caused a decrease in neutral red inclusion to 61% of the control on Day 7, with recovery to 86% on Day 12. Similarly, trypan blue exclusion decreased to 66% of the control following 7 days exposure to 40μg/ml FK506, and confluency of the monolayer was reduced to 50% as evidenced by phase contrast microscopy. After a 12-day exposure, treated monolayers became more confluent. On ultrastructural examination, FK506-treated cells exhibited increased cytoplasmic vacuolation and lipid inclusion. These data suggest that FK506 is reversibly and mildly toxic to monolayers of human renal proximal tubule cells and are consistent with clinical reports of reversible nephrotoxicity.     

FK506 enhanced osteoblastic differentiation in mesenchymal cells.

Bone morphogenetic protein (BMP) is a bone-derived growth factor capable of promoting the differentiation of mesenchymal cells into osteogenic lineage pathways. Recently, immunosuppressants were reported to cause a moderate increase in osteoblastic differentiation in a rat osteoblast-like osteosarcoma cell line. If immunosuppressants can induce osteoblastic differentiation, it will be useful for bone tissue transplantation. We assessed the effect of immunosuppressants with or without BMP-4 on inducing osteoblastic differentiation in osteoblast-like and other mesenchymal cells. FK506, an immunosuppressant often used clinically, induced a dose- and time-dependent increase in alkaline phosphatase (ALP) activity, one of the markers of osteoblast differentiation, in cells derived from mesenchyma. In the presence of BMP-4, ALP activity, mRNA levels of ALP and osteocalcin increased. FK506 was found to not only stimulate osteoblastic differentiation, but also to enhance BMP-4 induced osteoblastic differentiation. These results suggest that FK506 promotes differentiation of osteoblastic cells.     

Influences of Immunosuppressive Agents,FK506 and Cyclosporin on Systemic Candida albicans Infection in Mice

The effects of the immunosuppressive agents FK506 (tacrolimus) and cyclosporin (CyA) on Candida albicans infection in mice were compared with those of cyclophosphamide. FK506 and CyA did not exacerbate C. albicans infection in mice when the effects were determined on the basis of survival ratio and colony forming units (CFU) in the kidney, although cyclophosphamide (CY) impaired the host defence mechanisms of mice against C. albicans infection. The effects of FK506 and CyA on the body weight of mice, histopathological changes of lymphoid tissues and formation of granulomas in kidney were also studied in comparison with those of CY. This revised version was published online in June 2006 with corrections to the Cover Date.     

Effects of tacrolimus (FK506) on encephalomyocarditic virus-induced diabetes in mice

The effects of tacrolimus on insulin-dependent diabetes mellitus (IDDM) induced by the D-variant of encephalomyocarditis virus (D-EMCV) have been investigated. Male BALB/c mice were treated with tacrolimus before viral inoculation, and then were inoculated with 10 plaque forming units (PFU) of DEMCV. The mice continued to be treated with tacrolimus until the animals were sacrificed. D-EMCV-infected mice, which were treated with saline as controls, showed abnormal glucose tolerance test (GTT) values, whereas all infected mice with tacrolimus pretreatment were normal on 7 days-post inoculation (DPI). Histological observations revealed that non-treated tacrolimus D-EMCV-infected mice and which developed diabetes showed severe insulitis in their islets of Langerhans. On the other hand, D-EMCV-infected mice treated with tacrolimus were normal. In D-EMCV-infected mice, viruses in the pancreata were detected at the same level regardless of treatment with tacrolimus or saline. Expressions of TNF-alpha and IFN-gamma mRNA in spleens of tacrolimus-treated D-EMCV-infected mice were lower than that of non-treated tacrolimus DEMCV-infected mice on 7 DPI. The results suggest that tacrolimus suppresses expressions of TNF-alpha and IFN-gamma mRNAs to prevent the onset of D-EMCV-induced IDDM.     

Cyclosporine A- and FK506-induced apoptosis in PC12 cells

The purpose of this study was to examine, using glycogen synthase kinase (GSK) inhibitors, whether GSK-3 is involved in cyclosporine A (CsA)- and FK506-induced apoptosis in PC12 cells. CsA and FK506 increased apoptotic cell death with morphological changes characterized by cell shrinkage and nuclear condensation or fragmentation. Nerve growth factor (NGF) completely blocked cell death. Caspase-3 activation was accompanied by CsA- and FK506-induced cell death and inhibited by NGF. GSK-3 inhibitors such as alsterpaullone and SB216763 prevented CsA- and FK506-induced apoptosis. These results suggest that CsA and FK506 induce caspase-dependent apoptosis and that GSK-3 activation is involved in CsA- and FK506-induced apoptosis in PC12 cells.     

A calcineurin antifungal strategy with analogs of FK506

A novel antifungal strategy targeting the inhibition of calcineurin is described. To develop a calcineurin based inhibitor of pathogenic fungi, analogs of FK506 were synthesized that were able to permeate mammalian but not fungal cells. Antagonists in combination with FK506 were not immunosuppressive and retained antifungal activity in A. fumigatus. To reduce the dosage burden of the antagonist, murine oral PK was improved an order of magnitude relative to previous FK506 antagonists.     

Crystal structure of the FK506 binding domain of human FKBP25 in complex with FK506

Human FKBP25 (hFKBP25) is a nuclear immunophilin and interacts with several nuclear proteins, hence involving in many nuclear events. Similar to other FKBPs, FK506 binding domain (FKBD) of hFKBP25 also binds to immunosuppressive drugs such as rapamycin and FK506, albeit with a lower affinity for the latter. The molecular basis underlying this difference in affinity could not be addressed due to the lack of the crystal structure of hFKBD25 in complex with FK506. Here, we report the crystal structure of hFKBD25 in complex with FK506 determined at 1.8 Å resolution and its comparison with the hFKBD25–rapamycin complex, bringing out the microheterogeneity in the mode of interaction of these drugs, which could possibly explain the lower affinity for FK506.     

免疫抑制剂FK506对大鼠移植性肝癌肿瘤生长和脾转移的影响

目的观察在大鼠移植性肝癌模型中不同FK506剂量下肿瘤的大小和侵润以及脾脏转移情况,用以评价FK506对肝癌细胞增殖和转移的情况。方法在Wistar大鼠肝左叶种植walker-256细胞株成瘤的癌组织块以构建转移性肝癌模型,54只大鼠随机分为FK506低剂量组（0.5 mg/kg.d）,正常剂量组（1.0 mg/kg.d）和空白对照组,测量肿瘤大小,通过病理组织学观察7、14、21 d脾脏转移发生率和中位数。结果与空白组相比,第14、21天两个剂量组肝癌体积明显增大（P〈0.05）;同时也出现了脾转移,第14天FK506各组转移中位数均不同。根据病理组织学,各组脾转移的发生率没有明显性差异。结论用药早期,脾脏还存在免疫功能,免疫抑制剂不能完全压制抗肿瘤效应。低剂量FK506也许有利于减少癌症脾转移,仍需进一步的探讨FK506低剂量长期使用在肝癌复发和转移的作用。     

Microbial transformation of immunosuppressive compounds III. Glucosylation of immunomycin (FR 900520) and FK 506 byBacillus subtilis ATCC 55060

Summary The regiospecific glucosylation of FK 506 and immunomycin (FR 900520) at the 24-hydroxy position was performed using resting cells ofBacillus subtilis ATCC 55060. 24-Glucopyranosyl FK 506 and 24-glucopyranosyl immunomycin were isolated by methylene chloride extraction and purification using reverse phase HPLC. The metabolite structures were established using spectroscopic techniques including MS and NMR. The glucose conjugate was further confirmed by chemical degradation. Enzymatic glucosylation was demonstrated using cell-free extracts derived fromBacillus subtilis ATCC 55060. The 24-glucosyltransferase, which appears UDP-glucose dependent, was solubilized from cell membranes by treatment with 0.1% Nonidet P-40 detergent. The optimal conditions for assay of the enzyme have been determined.     

Neuroprotectant FK506 inhibits glutamate-induced apoptosis of astrocytes in vitro and in vivo

Neuron-astrocyte interactions are critical for signalling, energy metabolism, extracellular ion and glutamate homeostasis, volume regulation and neuroprotection in the CNS. Glutamate uptake by astrocytes may prevent excitotoxic glutamate elevation and determine neuronal survival. However, an excess of glutamate can cause the death of astrocytes. FK506, an inhibitor of calcineurin, and an immunosuppressive drug, is neuroprotective in animal models of neurologic diseases, including focal and global ischaemia. In the present work, we demonstrate that a single injection of FK506 60 min after a transient middle cerebral artery occlusion (MCAo) significantly decreases the number of terminal deoxynucleotidyl transferase nick-end labelling (TUNEL)-positive cells in the ischaemic cortex and striatum. Using 3-D confocal microscopy we found that, 24 h after MCAo, many TUNEL-positive cells in the ischaemic striatum and cortex are astrocytes. Furthermore, we demonstrate that exposure of cultured cortical astrocytes to 50-100 mM Glu for 24 h induces apoptotic alterations in nuclear morphology, DNA fragmentation, dissipation of mitochondrial transmembrane potential (DeltaPsi) and caspase activation. FK506 (1 muM) efficiently inhibits Glu-induced apoptosis of cultured astrocytes, DNA fragmentation and changes in mitochondrial DeltaPsi. Our findings suggest that modulation of glutamate-induced astrocyte death early after reperfusion may be a novel mechanism of FK506-mediated neuroprotection in ischaemia.     

Effect of FK506 on osteoinduction by recombinant human bone morphogenetic protein-2

FK506 is an immunosuppressant that is used widely in organ transplantation, and it has recently been recognized as effective for promoting the growth of bone grafts [J. Bone Miner. Res. 15 (2000) 1147]. In this study, we evaluated the influence of FK506 on osteoinduction by recombinant human bone morphogenetic protein-2 (rhBMP-2) using atelopeptide type I collagen as a carrier. We administered FK506 (1 mg/kg/day intramuscularly) on days -2 to 0, -2 to 7, and -2 to sacrifice. rhBMP-2 was implanted into the calf muscle of Wistar rats (thirty per group) and the implant was sampled on days 7, 14, and 21. Radiographic evaluation, histological examination, and biochemical analysis were performed. It was found that FK506 promoted the early stage of osteoinduction after short-term administration. However, long-term administration of this agent accelerated both bone formation and bone resorption. In order to use FK506 effectively for promoting bone growth, we must further examine the appropriate dose, method, and period of administration.     

Liquid chromatography-mass spectrometry characterization of FK506 biosynthetic intermediates in Streptomyces clavuligerus KCTC 10561BP

The development of an efficient analytical method for the reliable detection and identification of the biosynthetic intermediates found in microbial cultures, which usually produce complex intermediates of the metabolites of interest, is essential for further biosynthetic investigations. This study developed a simple and highly selective method for detecting the biosynthetic intermediates involved in the FK506 pathway of Streptomyces clavuligerus KCTC 10561BP involving a cleanup procedure using a solid-phase extraction technique to provide reliable extraction of FK506-related compounds from a cell culture broth and liquid chromatography-electrospray ionization-tandem mass spectrometry (LC-ESI-MS/MS) to separate and detect the FK506-related intermediates at concentrations as low as 0.2 μg/L in the broth. This method enabled the analytical profiling of the intermediates formed during the biosynthesis of FK506 in this S. clavuligerus strain, which produced FK506 as a main product. Eight FK506 intermediates—FK520, 37,38-dihydroFK506, prolylFK506, 9-decarbonyl-9-hydroxylFK506, 9-deoxoFK506, desmethylFK520, prolylFK520, and 9-deoxoFK520—were identified. This is the first report of the LC-ESI-MS/MS characterization of a wide range of FK506 analogs from a bacterial fermentation broth. The protocol employed in this study may be useful for estimating the structure of the metabolites without the need for a time-consuming isolation process and nuclear magnetic resonance (NMR) spectroscopy.     

Effect of FK506 in reducing scar formation by inducing fibroblast apoptosis after sciatic nerve injury in rats

We previously demonstrated that FK506, a generally applied immunosuppressant in organ transplantation, could promote peripheral nerve regeneration through reducing scar formation. However, little is known about how FK506 reduces scar formation. Herein we investigated the influence of FK506 on fibroblast proliferation and its correlation with scar formation after sciatic nerve injury in rats, and further explored the effect of FK506 on fibroblast proliferation and apoptosis in vitro. Masson staining and immunohistochemistry revealed that scar area and fibroblast number in the nerve anastomosis of sciatic nerve-injured rats were significantly reduced after FK506 administration. The scar area had a significant positive correlation with the fibroblast number, as detected by linear correlation analysis. CCK-8 assay and flow cytometry indicated that FK506 also inhibited proliferation and induced apoptosis of fibroblasts in vitro. It was primarily phosphorylation of JNK and ERK that were activated during the apoptosis of fibroblast. Pretreatment of cells with JNK inhibitor, SP600125, or ERK inhibitor, PD98059, could inhibit FK506-induced fibroblast apoptosis, respectively. Moreover, simultaneous application of both inhibitors had additive roles in cell protection from apoptosis. These results suggest that FK506-induced fibroblast apoptosis contributes to the suppression of fibroblast proliferation and then results in the reduction of scar formation in sciatic nerve-injured rat, and that JNK and ERK are involved in FK506-induced fibroblast apoptosis.     

FK506 reduces abnormal prion protein through the activation of autolysosomal degradation and prolongs survival in prion-infected mice

Prion diseases are fatal neurodegenerative disorders and no effective treatment has been established to date. In this study, we evaluated the effect of FK506 (tacrolimus), a macrolide that is known to be a mild immunosuppressant, on prion infection, using cell culture and animal models. We found that FK506 markedly reduced the abnormal form of prion protein (PRNP^Sc) in the cell cultures (N2a58 and MG20) infected with Fukuoka-1 prion. The levels of autophagy-related molecules such as LC3-II, ATG12–ATG5 and ATG7 were significantly increased in the FK506-treated cells, and resulted in the increased formation of autolysosomes. Upregulation of the autophagy-related molecules was also seen in the brains of FK506-treated mice and the accumulation of PRNP^Sc was delayed. The survival periods in mice inoculated with Fukuoka-1 were significantly increased when FK506 was administered from day 20 post-inoculation. These findings provide evidence that FK506 could constitute a novel antiprion drug, capable of enhancing the degradation of PRNP^Sc in addition to attenuation of microgliosis and neuroprotection.     

Co-stimulation with bone morphogenetic protein-9 and FK506 induces remarkable osteoblastic differentiation in rat dedifferentiated fat cells

Dedifferentiated fat (DFAT) cells, which are isolated from mature adipocytes using the ceiling culture method, exhibit similar characteristics to mesenchymal stem cells, and possess adipogenic, osteogenic, chondrogenic, and myogenic potentials. Bone morphogenetic protein (BMP)-2 and -9, members of the transforming growth factor-β superfamily, exhibit the most potent osteogenic activity of this growth factor family. However, the effects of BMP-2 and BMP-9 on the osteogenic differentiation of DFAT remain unknown. Here, we examined the effects of BMP-2 and BMP-9 on osteoblastic differentiation of rat DFAT (rDFAT) cells in the presence or absence of FK506, an immunosuppressive agent. Co-stimulation with BMP-9 and FK506 induced gene expression of runx2, osterix, and bone sialoprotein, and ALP activity compared with BMP-9 alone, BMP-2 alone and BMP-2 + FK506 in rDFAT cells. Furthermore, it caused mineralization of cultures and phosphorylation of smad1/5/8, compared with BMP-9 alone. The ALP activity induced by BMP-9 + FK506 was not influenced by addition of noggin, a BMP antagonist. Our data suggest that the combination of BMP-9 and FK506 potently induces osteoblastic differentiation of rDFAT cells.     

Combination therapy with transductive anti-death FNK protein and FK506 ameliorates brain damage with focal transient ischemia in rat

Many practical therapies have been explored as clinical applications for ischemic cerebral infarction; however, most are still insufficient to treat acute stroke. We show here a potential combination therapy in a rat focal ischemic model to improve neurological symptoms as well as to reduce infarct volumes at the maximum level. We applied protein transduction technology using artificial anti-death Bcl- x _l derivative with three amino acid-substitutions (Y22 F , Q26 N and R165 K ) (FNK) protein fused with a protein-transduction-domain peptide (PTD-FNK). When PTD-FNK was administrated 1 h after initiating ischemia followed by the administration of an immunosuppressant FK506 with a 30-min time lag, infarct volumes of the total brain and cortex were markedly reduced to 27% and 14%, respectively. This procedure not only reduced the infarct volume and edema, but also markedly improved neurological symptoms. The therapeutic effect continued for at least 1 week after ischemia. FK506 inhibited the transduction of PTD-FNK in vitro , which explains the requirement of a time lag for the administration of FK506. An additional in vitro experiment showed that PTD-FNK, when administered 30 min before FK506, gave the maximal protective effect by reducing the intracellular calcium concentration. We propose that this combination therapy would provide a synergistic protective effect by both drugs, reducing adverse the effects of FK506.     

FK506 binding protein 12 differentially accelerates fibril formation of wild type alpha-synuclein and its clinical mutants A30P or A53T

Aggregation of alpha-synuclein (α-SYN) plays a key role in Parkinson's disease. We have previously shown that aggregation of α-SYN in vitro is accelerated by addition of FK506 binding proteins (FKBP) and that this effect can be counteracted by FK506, a specific inhibitor of these enzymes. In this paper, we investigated in detail the effect of FKBP12 on early aggregation and on fibril formation of wild-type, A53T and A30P α-SYN. FKBP12 has a much smaller effect on the fibril formation of these two clinical mutants α-SYN. Using an inactive enzyme, we were able to discriminate between catalytic and non-catalytic effects that differentially influence the two processes. A model explaining non-linear concentration dependencies is proposed.     

Specific inhibition of hepatitis C virus replication by cyclosporin A

The difficulty in eradicating hepatitis C virus (HCV) infection is attributable to the limited treatment options against the virus. Recently, cyclosporin A (CsA), a widely used immunosuppressive drug, has been reported to be effective against HCV infection [J. Gastroenterol. 38 (2003) 567], although little is understood about the mechanism of its action against HCV. In this study, we investigated the anti-viral effects of CsA using an HCV replicon system. Human hepatoma Huh7 cells were transfected with an HCV replicon expressing a chimeric gene encoding a luciferase reporter and neomycin phosphotransferase (Huh7/Rep-Feo). Treatment of the Huh7/Rep-Feo cells with CsA resulted in suppression of the replication of the HCV replicon in a dose-dependent manner, with an IC50 of approximately 0.5 microg/ml. There were no changes in the rate of cell growth or viability, suggesting that the effect of CsA against HCV is specific and not due to cytotoxicity. In contrast, FK506, another immunosuppressive drug, did not suppress HCV replication. CsA did not activate interferon-stimulated gene responses, suggesting that its action is independent of that of interferon. In conclusion, CsA inhibits HCV replication in vitro specifically at clinical concentrations. Further defining its mode of action against HCV replication potentially may be important for identifying novel molecular targets to treat HCV infection.