FKBPL and its peptide derivatives inhibit endocrine therapy resistant cancer stem cells and breast cancer metastasis by downregulating DLL4 and Notch4.

BACKGROUND: Optimising breast cancer treatment remains a challenge. Resistance to therapy is a major problem in both ER- and ER+ breast cancer. Tumour recurrence after chemotherapy and/or targeted therapy leads to more aggressive tumours with enhanced metastatic ability. Self-renewing cancer stem cells (CSCs) have been implicated in treatment resistance, recurrence and the development of metastatic disease. METHODS: In this study, we utilised in vitro, in vivo and ex vivo breast cancer models using ER+ MCF-7 and ER- MDA-MB-231 cells, as well as solid and metastatic breast cancer patient samples, to interrogate the effects of FKBPL and its peptide therapeutics on metastasis, endocrine therapy resistant CSCs and DLL4 and Notch4 expression. The effects of FKBPL overexpression or peptide treatment were assessed using a t-test or one-way ANOVA with Dunnett's multiple comparison test. RESULTS: We demonstrated that FKBPL overexpression or treatment with FKBPL-based therapeutics (AD-01, pre-clinical peptide /ALM201, clinical peptide) inhibit i) CSCs in both ER+ and ER- breast cancer, ii) cancer metastasis in a triple negative breast cancer metastasis model and iii) endocrine therapy resistant CSCs in ER+ breast cancer, via modulation of the DLL4 and Notch4 protein and/or mRNA expression. AD-01 was effective at reducing triple negative MDA-MB-231 breast cancer cell migration (n ≥ 3, p < 0.05) and invasion (n ≥ 3, p < 0.001) and this was translated in vivo where AD-01 inhibited breast cancer metastasis in MDA-MB-231-lucD3H1 in vivo model (p < 0.05). In ER+ MCF-7 cells and primary breast tumour samples, we demonstrated that ALM201 inhibits endocrine therapy resistant mammospheres, representative of CSC content (n ≥ 3, p < 0.05). Whilst an in vivo limiting dilution assay, using SCID mice, demonstrated that ALM201 alone or in combination with tamoxifen was very effective at delaying tumour recurrence by 12 (p < 0.05) or 21 days (p < 0.001), respectively, by reducing the number of CSCs. The potential mechanism of action, in addition to CD44, involves downregulation of DLL4 and Notch4. CONCLUSION: This study demonstrates, for the first time, the pre-clinical activity of novel systemic anti-cancer therapeutic peptides, ALM201 and AD-01, in the metastatic setting, and highlights their impact on endocrine therapy resistant CSCs; both areas of unmet clinical need.

GM1485, a nonimmunosuppressive immunophilin ligand, promotes neurofunctional improvement and neural regeneration following stroke.

Stroke is the leading cause of disability in the industrialized world, and the development of pharmacologic strategies to promote poststroke recovery is of paramount importance. GM1485, a nonimmunosuppressive immunophilin ligand, promotes regeneration of multiple cell types following injury. In the present study, we evaluated the effect of GM1485 treatment on functional recovery and neurogenesis following rat stroke. Transient cerebral ischemia was induced in rats receiving daily GM1485 (5 mg/kg) beginning 24 hr postischemia and continuing for a total of 6 weeks. Neurological function was evaluated over this period using a battery of neurobehavioral tests, and immunostaining for stem-cell markers was performed following animal sacrifice. An in vitro model of oxidative stress was also employed to evaluate the ability of GM1485 to mediate stem-cell-like induction and plasticity. GM1485-treated rats demonstrated improved neurological function as well as increased Sox2(+) cells in the ipsilateral SVZ and striatum relative to vehicle-treated rats. Additionally, GM1485-treated fibroblasts subjected to oxidative stress were reprogrammed to a stem-cell-like phenotype and were able to differentiate down a neuronal lineage. These data demonstrate that GM1485 administration improves neurological function and is consistent with an upregulation of endogenous neurogenesis following stroke in rats. Further experiments are necessary to characterize the molecular pathways involved in these processes.

The therapeutic and diagnostic potential of FKBPL; a novel anticancer protein.

The immunophilin family of proteins has a vast number of roles regulating a variety of biological processes through protein-protein interactions. A relatively new and divergent member of this family, FK506-binding protein like (FKBPL), is emerging as a key player in the DNA damage response, steroid receptor signalling and more recently, control of tumour growth where it regulates response to endocrine therapy in addition to acting as a novel antiangiogenic protein. As a new therapeutic peptide based on FKBPL approaches clinical trials, this article highlights a unique approach to targeting tumours that are resistant to current antiangiogenic therapies and supports the role of FKBPL as a novel prognostic and predictive biomarker, distinct from its other family members.

FKBPL and peptide derivatives: novel biological agents that inhibit angiogenesis by a CD44-dependent mechanism.

PURPOSE: Antiangiogenic therapies can be an important adjunct to the management of many malignancies. Here we investigated a novel protein, FKBPL, and peptide derivative for their antiangiogenic activity and mechanism of action. EXPERIMENTAL DESIGN: Recombinant FKBPL (rFKBPL) and its peptide derivative were assessed in a range of human microvascular endothelial cell (HMEC-1) assays in vitro. Their ability to inhibit proliferation, migration, and Matrigel-dependent tubule formation was determined. They were further evaluated in an ex vivo rat model of neovascularization and in two in vivo mouse models of angiogenesis, that is, the sponge implantation and the intravital microscopy models. Antitumor efficacy was determined in two human tumor xenograft models grown in severe compromised immunodeficient (SCID) mice. Finally, the dependence of peptide on CD44 was determined using a CD44-targeted siRNA approach or in cell lines of differing CD44 status. RESULTS: rFKBPL inhibited endothelial cell migration, tubule formation, and microvessel formation in vitro and in vivo. The region responsible for FKBPL's antiangiogenic activity was identified, and a 24-amino acid peptide (AD-01) spanning this sequence was synthesized. It was potently antiangiogenic and inhibited growth in two human tumor xenograft models (DU145 and MDA-231) when administered systemically, either on its own or in combination with docetaxel. The antiangiogenic activity of FKBPL and AD-01 was dependent on the cell-surface receptor CD44, and signaling downstream of this receptor promoted an antimigratory phenotype. CONCLUSION: FKBPL and its peptide derivative AD-01 have potent antiangiogenic activity. Thus, these agents offer the potential of an attractive new approach to antiangiogenic therapy.

Diabetic neuropathy: therapies on the horizon.

OBJECTIVES: This is a review of emerging interventions from the recent preclinical and clinical literature that demonstrate the potential for effectiveness in the therapy of diabetic neuropathy (DN). DN is the most common complication of diabetes mellitus and up to 50% of patients with type 1 and type 2 forms have some or other form of neuropathy. The pathology of DN is characterized by progressive nerve fibre loss that gives rise to positive and negative clinical signs and symptoms such as pain, paraesthesiae and loss of sensation. KEY FINDINGS: There are very few drugs available to directly treat DN. Those that are clinically indicated provide symptomatic relief but do not repair or reverse underlying nerve damage. However, some agents are in clinical development that may support adult neurons and direct reparative processes after injury stages. Several disease modifying drugs such as aldose reductase inhibitors and protein kinase C inhibitors are in phase III development. Agents on the horizon include neurotrophic factors, growth factors, gene therapy, immunotherapy, poly(ADP-ribose) polymerase inhibitors and non-immunosuppressive immunophilin ligands. SUMMARY: Progress has been made toward understanding the biochemical mechanisms leading to diabetic neuropathy, and as a result, new treatment modalities are being explored. The pathogenesis, types and approaches for treating DN together with the newer therapeutic interventions on the horizon are discussed.

Protective effects of immunophilin ligands on testicular torsion/detorsion damage in rats.

OBJECTIVES: The purpose was to investigate the role of immunophilin ligands in ischemia/reperfusion (I/R)-induced germ cell apoptosis in the rat. MATERIALS AND METHODS: Sprague-Dawley rats were divided into five groups with ten animals in each. In animals undergoing torsion/detorsion, right testes were rotated 720 degrees for 1 h. A baseline group was for basal normal values. The sham-operated group served as a control group. The TD group underwent torsion/detorsion surgery alone; the cyclosporine-A group (TD-CsA) received intravenous cyclosporine injection (5 mg/kg) at the time of detorsion, and the FK-506 group (TD-FK) received intravenous FK-506 (3.5 mg/kg) at the time of detorsion. For measurement of lipid peroxidation and antioxidant enzyme activities, the right testes of five animals in each group were excised after 4-h reperfusion. Germ cell apoptosis indices were determined 24 h following detorsion in the right testes of the remaining five animals in each group. RESULTS: Malondialdehyde (MDA) levels in the TD group were significantly higher compared to control and baseline groups. Moreover, testicular MDA values in TD-CsA and TD-FK groups were significantly lower than in TD. There were also significant decreases in catalase and superxide dismutase activities in the TD group compared to control and baseline groups. These values in TD-CsA and TD-FK groups were significantly higher than in TD. The mean germ cell apoptosis scores were significantly higher in TD animals compared to control and baseline groups; however, CsA and FK-506 treatment significantly reduced the apoptosis compared with the TD group. CONCLUSION: We have shown that administration of immunophilin ligands in testicular torsion decreases ischemia/reperfusion (I/R) cellular damage. The results of biochemical studies suggest that reduction of oxidative stress along with attenuated neutrophil accumulation by immunophilin ligands may have a major role in their cytoprotective effects.

Neuroprotective and antiretroviral effects of the immunophilin ligand GPI 1046.

HIV infection results in a neurodegenerative disorder for which currently there is no effective therapy available. Currently, available antiretroviral therapy has no impact on the production of early regulatory HIV proteins once the virus is integrated. Of these proteins, Tat was shown to be toxic to neurons. We, thus, used an in vitro neuronal culture system to determine if immunophilin ligands could protect against Tat-induced neurotoxicity. We found that GPI 1046 had potent neuroprotective effects in this model. The compound was able to protect the neurons even though it only partially obliterated Tat-induced oxidative stress in neurons, suggesting that other mechanisms may be important in mediating its neuroprotective effect. Furthermore, GPI 1046 showed inhibition of HIV replication and Tat-mediated long terminal repeat (LTR) activation suggesting that this class of compounds may be worthy of further exploration as a potential treatment for HIV dementia.

Therapeutic potential of natural product signal transduction agents.

Modern drug discovery embraces a strategy of targeting cellular signal transduction pathways as a means of finding new therapeutic agents. Historically, natural products derived from microorganisms have played an important role as drug leads and clinical candidates under this paradigm. The future drug potential of natural products as signal transduction agents looks promising, as illustrated by two key examples. First, substantial advances have been made in the development of inhibitors based on immunophilin ligand polyketides, which target the TOR-mediated pathways and can modulate processes including cell proliferation and cell-cycle arrest. Second, the discovery of natural product inhibitors of the ubiquitin-proteasome proteolytic signal transduction pathway represents an emerging field. Given these examples, together with the diversity of as yet undiscovered agents, natural product signal transduction agents offer great potential for future drug discovery efforts.

Neuroprotection in the PNS: erythropoietin and immunophilin ligands.

Many illnesses that affect the peripheral nervous system (PNS) lead to distal axonal degeneration rather than loss of neuronal cell bodies. Strategies aimed at promoting survival of injured neurons (i.e., preventing cell death) may not be applicable to many PNS illnesses. We have developed in vitro and in vivo animal models to study mechanisms of acquired peripheral neuropathies and used these models to evaluate the therapeutic potential of novel compounds. In recent years, erythropoietin (EPO) has been recognized as a novel neuroprotectant in the central nervous system. In the PNS, we recently showed that Schwann cell-derived EPO acts as an endogenous neuroprotectant and that it is most effective in preventing distal axonal degeneration seen in models of peripheral neuropathy. Similarly, we showed that immunophilin ligands are also neuroprotective in the PNS and prevent axonal degeneration seen in models of peripheral neuropathies. Both EPO and non-immunosuppressive immunophilin ligands are in early clinical development for the treatment of acquired peripheral neuropathies.

[Inhibitors of NFAT-calcineurin pathway].

Cyclosporin A(CsA) and tacrolimus (FK506) are important immunosuppressants to inhibit rejection of transplanted organs and to treat various immunological disorders, however those drugs produce major side effects. Those drugs form complexes with cellular proteins, immunophilins (cyclophilin for CsA and FKBP for tacrolimus) and inhibit Ca-calmodulin dependent phosphatase calcineurin through direct binding. Calcineurin dephosphorylates various substrates including NFAT family proteins required for the expression of immunoregulatory molecules especially cytokines. NFAT-calcineurin pathway offers a good model system to apply new technology to develop drugs. Enzyme-substrate interaction could be an important target to develop drugs with high specificity accompanied with less side effects.

Immunosuppressants: neuroprotection and promoting neurological recovery following peripheral nerve and spinal cord lesions.

No clinical techniques induce restoration of neurological losses following spinal cord trauma. Peripheral nerve damage also leads to permanent neurological deficits, but neurological recovery can be relatively good, especially if the ends of a transected nerve are anastomosed soon after the injury. The time until recovery generally depends on the distance the axons must regenerate to their targets. Neurological recovery following the destruction of a length of a peripheral nerve requires a graft to bridge the gap that is permissive to, and promotes, axon regeneration. But neurological recovery is slow and limited, especially for gaps longer than 1.5 cm, even using autologous peripheral nerve grafts. Without a reliable means of bridging long nerve gaps, such injuries commonly result in amputations. Promoting extensive neurological recovery requires techniques that simultaneously provide protection to injured neurons and increase the numbers of neurons that extend axons, while inducing more rapid and extensive axon regeneration across long nerve gaps. Although conduits filled with various materials enhance axon regeneration across short nerve gaps, pure sensory nerve graft remains the gold standard for use across long nerve gaps, even though they lead to only limited neurological recovery. Consistent results demonstrate that several immunosuppressive agents enhance the number of axons and the rate at which they regenerate. This review examines the roles played by immunosuppressants, especially FK506, with primary focus on its role as a neuroprotectant and neurotrophic agent, and its potential clinical use to promote improved neurological recovery following peripheral nerve and spinal cord injuries.

Co-expression and immunity of Legionella pneumophila mip gene and immunoadjuvant ctxB gene.

The mip gene of Legionella pneumophila and the ctxB gene of Vibrio cholerae were amplified by PCR respectively. The amplified cDNA was ligated to the pcDNA3.1(+) vector. The recombinant plasmids pcDNA3.1-mip and pcDNA3.1-ctxB were identified by restriction analysis and PCR, and further confirmed by sequencing analysis. NIH3T3 cells were transfected with pcDNA3.1-mip and pcDNA3.1-ctxB according to the Lipofection method. Transient and stable products of the co-expression of the mip gene and ctxB gene were detected by immunofluorescence and Western blotting. The results showed that NIH3T3 cells were successfully transfected, and that the transiently and stably co-expressed products can be detected in the transfected cells. To detect the humoral and cellular immune response in immunized mice induced by the co-mmunization of the mip and ctxB genes, female BALB/c mice were immunized intramuscularly with pcDNA3.1-mip and pcDNA3.1-ctxB. The results showed that the specific antibody titer and the cytotoxic T-lymphocyte response for pcDNA3.1-mip immunization and co-immunization were increased compared with that of pcDNA3.1(+) immunization. Furthermore, the specific antibody titer and cytotoxic T-lymphocyte response for co-immunization were increased compared with that of pcDNA3.1-mip immunization. Statistical analysis using one-way analysis of variance (ANOVA) showed that there was a significant difference between the groups (P<0.01). The results indicated that the ctxB gene enhanced the humoral and cellular immune response to the mip gene immunization. These findings provide experimental evidence to support the development of the L. pneumophila DNA vaccine.

Current and future immunosuppressive strategies in renal transplantation.

The past decade has witnessed the introduction of several new immunosuppressive agents. The availability of these new pharmacologic offerings has not diminished the challenge of achieving a balance of adequate graft protection while minimizing the consequences of excessive immunosuppression. For renal transplant recipients, maintenance immunosuppression generally consists of a calcineurin inhibitor in combination with an antiproliferative agent and a corticosteroid; more recently, mammalian target of rapamycin inhibitors have been used. Excellent results have been achieved at many transplant centers with combinations of these agents in a variety of protocols. Regimens designed to limit or eliminate calcineurin inhibitor and/or corticosteroid therapy are actively being pursued in the transplant community. Allograft tolerance and xenotransplantation are being studied, and the knowledge gained from the effort may help in the development of innovative strategies and new immunosuppressive agents.

FK506 and a nonimmunosuppressant derivative reduce axonal and myelin damage in experimental autoimmune encephalomyelitis: neuroimmunophilin ligand-mediated neuroprotection in a model of multiple sclerosis.

Multiple sclerosis (MS) is an inflammatory disease of the central nervous system (CNS) in which demyelination and axonal loss result in permanent neurologic disability. We examined the neuroprotective property of the immunosuppressant FK506 (tacrolimus), FK1706 (a nonimmunosuppressant FK506 derivative) and cyclosporin A (CsA) in a chronic relapsing experimental autoimmune encephalomyelitis (EAE) model of MS. Female SJL/J mice were immunized by subcutaneous (s.c.) injection with proteolipid protein 139-151 peptide in complete Freund's adjuvant. At the onset of paralysis, 12-14 days after immunization, mice received daily s.c. injections of FK506 (0.2, 1, and 5 mg/kg), FK1706 (5 mg/kg), CsA (2, 10, and 50 mg/kg), saline or vehicle (30% dimethylsulfoxide) for 30 days. FK506 (at a dose of 5 mg/kg) reduced the severity of the initial disease and suppressed relapses. FK1706 did not significantly alter the clinical course and CsA (at a dose of 50 mg/kg) lessened the severity of the initial episode of EAE but did not alter relapses. In the thoracic spinal cord, FK506 (5 mg/kg), FK1706 (5 mg/kg), and CsA (50 mg/kg) significantly (P < 0.001) reduced the extent of damage in the dorsal, lateral, and ventral white matter by a mean of up to 95, 68, and 30%, respectively. A nonimmunosuppressant dose of FK506 (0.2 mg/kg) also significantly (P < 0.001) reduced the extent of damage in the spinal cord by a mean of up to 45%. Other dosages of these compounds were ineffective. FK506 markedly protects against demyelination and axonal loss in this MS model through immunosuppression and neuroprotection.

Neuroregenerative and neuroprotective actions of neuroimmunophilin compounds in traumatic and inflammatory neuropathies.

FK506 (tacrolimus, Prograf is an immunosuppressant drug that also has profound neuroregenerative and neuroprotective actions independent of its immunosuppressant activity. The separation of these properties has led to the development of non-immunosuppressant derivatives that retain the neurotrophic activity. This review focuses on the peripheral nerve actions of these compounds following mechanical injury (nerve crush or transection with graft repair) and in models of inflammatory neuropathies. Whereas FK506 may be indicative for the treatment of inflammatory neuropathies where its immunosuppressive action would be advantageous, non-immunosuppressant derivatives represent a new class of potential therapeutic agents for the treatment of human neurological conditions in general. Moreover, these studies have led to the discovery of a novel mechanism whereby these compounds activate intrinsic neuroregenerative and neuroprotective pathways in the neuron.

[Immunophilin ligands: from immunosuppressants to neuroprotective drugs].

Non-immunosuppressive immunophilin ligands (NI-IPLs) are attracting attention as new candidate drugs for neuroprotection and/or neurorestoration, particularly since they do not have the adverse effects of immunosuppressants. However, it is not yet enough to understand that NI-IPLs are useful drugs for treating neurological disorders. In particular, the molecular mechanism of NI-IPL activity in target cells in the brain remains obscure. In this review, we focused on the molecular basis of the neuroprotective properties of IPLs. Our findings suggest that IPLs have neuroprotective effects mediated by multiple beneficial properties such as a glutathione (GSH)-activating effect, a neurotrophic factor (NTF)-activating effect, and an anti-apoptotic effect, but not by an immunosuppressive effect, both in cell cultures and in vivo. In particular, the GSH-activating effect and the NTF-activating effect of NI-IPLs may be essential to the expression of their neuroprotective properties. Thus, NI-IPLs might have a potentially beneficial effect by ameliorating neurological disorders, since they do not cause serious side effects such as immune deficiency.

Therapeutic implications for immunophilin ligands in the treatment of neurodegenerative diseases.

There is a significant unmet need for therapeutic agents in the treatment of neurodegenerative diseases. Given their clinical importance, prototypical molecules that clearly exhibit both neuroprotective and neuroregenerative activities have been highly sought after. The journey led to the exploitation of neurotrophins, a family of proteins that had extraordinary therapeutic properties in pre-clinical models of neurodegeneration. Although experimentally promising, clinical development of neurotrophins for various neurological indications, such as Alzheimer's Disease, Amyotrophic Lateral Sclerosis, and Parkinson's Disease was met with severe obstacles and setbacks, such as the inability to deliver these large proteins to target population of neurons, instability of the proteins, and non-specific activity. Immunophilins are proteins that act as receptors for immunosuppresant drugs, i.e. FK506 (tacrolimus), cyclosporin A, and rapamycin (sirolimus, Rapamune). Studies indicate immunophilins are expressed 10-100 fold higher in CNS and PNS tissue than in immune tissue. Subsequent studies revealed potent neuroprotective and neuroregenerative properties of immunophilin ligands in both culture and animal models. In contrast to neurotrophins, most immunophilin ligands are highly stable, small molecules that can readily cross the blood-brain barrier and are orally bioavailable. Taken together, these data prompted the development of nonimmunosuppressive immunophilin ligands with potent therapeutic activities, although the potency of select compounds has come into question in more recent studies. This review will examine the experimental evidence supporting the use of immunophilin ligands for the treatment of neurodegenerative diseases and the current progression of these molecules in clinical trials.

Regulation of MAPK signaling pathways through immunophilin-ligand complex.

It is well established that the immunosuppressive effects of cyclosporin A (CsA) and FK506 (also known as tacrolimus) are mediated through binding to their cognate cellular proteins cyclophilin and FKBP (collectively termed immunophilins), respectively. Biochemical analysis had revealed that cyclophilin-CsA and FKBP-FK506 complexes bind to and inactivate Ca(2+)-dependent serine/threonine phosphatase calcineurin. Since calcineurin regulates nuclear translocation and subsequent activation of nuclear factor of activated T cells (NFAT) transcription factors that is one of essential steps for cytokine gene expression in activated T cells, it is believed that inhibition of calcineurin is a molecular basis of the immunosuppressive properties of CsA and FK506. However, recent studies indicate that both CsA and FK506 can block activation of JNK and p38 signaling pathways during T cell activation. CsA and FK506, thus, have two distinct mechanisms of action; one is the inhibition of the protein phosphatase activity of calcineurin, leading to the blockade of the nuclear translocation of NFAT transcription factors, and the other is the suppression of JNK and p38 activation pathways. It is likely that the presence of two distinct targets in T cell activation makes CsA and FK506 highly potent immunosuppressive drugs. Here we discuss the action of immunophilin-ligand complexes on JNK and p38 activation pathways. We also argue the possibility of immunotherapeutic application targeting at JNK and p38 signaling pathways.