Supplementary Figures 1-10 from FTY720 (Fingolimod) Sensitizes Prostate Cancer Cells to Radiotherapy by Inhibition of Sphingosine Kinase-1

Supplementary Figures 11-14 from FTY720 (Fingolimod) Sensitizes Prostate Cancer Cells to Radiotherapy by Inhibition of Sphingosine Kinase-1

Background. High mortality of haemodialysis patients is associated with systemic chronic inflammation and overactivation of the renin-angiotensin system (RAS). Insufficient elimination of pro-inflammatory immune mediators, especially in the molecular weight range of 15-45 kDa, may be one of the reasons for this. Employment of haemodialysis membranes with increased permeability was shown to ameliorate the inflammatory response and might modulate the effects of local RAS. In this study, we tested the impact of high cut-off (HCO), medium cut-off (MCO) and high-flux (HF) dialysis on leucocytic transcripts of angiotensin-converting enzymes (ACE and ACE2). Additionally, the impact of HCO, MCO and HF sera and dialysates on local ACEs and inflammation markers was tested in THP-1 monocytes. Methods. Patients' leucocytes were obtained from our recent clinical studies comparing HCO and MCO dialysers with HF. The cells were subjected to quantitaive polymerase chain reaction (qPCR) analyses with TaqMan probes specific for ACE, ACE2 and angiotensin II (AngII) and Ang1-7 receptors. Sera and dialysates from the clinical trials as well as samples from in vitro dialysis were tested on THP-1 monocytic cells. The cells were subjected to qPCR analyses with TaqMan probes specific for ACE, ACE2, interleukin-6 and tumour necrosis factor a and immunocytochemistry with ACE and ACE2 antibodies. Results. Leucocytes obtained from patients treated with HCO or MCO demonstrated decreased transcript expression of ACE, while ACE2 was significantly upregulated as compared with HF. Receptors for AngII and Ang1-7 remained unchanged. THP-1 monocytes preconditioned with HCO and MCO patients' or in vitro dialysis sera reflected the same expressional regulation of ACE and ACE2 as those observed in HCO and MCO leucocytes. As a complementary finding, treatment with HCO and MCO in vitro dialysates induced a pro-inflammatory response of the cells as demonstrated by elevated messenger RNA expression of tumour necrosis factor a and interleukin-6, as well as upregulation of ACE and decreased levels of ACE2. Conclusions. Taken together, these data demonstrate that employment of membranes with high permeability eliminates a spectrum of mediators from circulation that affect the RAS components in leucocytes, especially ACE/ACE2.

Resistance to docetaxel is a key problem in current prostate and breast cancer management. We have recently discovered a new molecular mechanism of prostate cancer docetaxel chemoresistance mediated by the mammalian target of rapamycin (mTOR)/sphingosine-kinase-1 (SK1) pathway. Here we investigated the influence of this pathway on vascular endothelial growth factor (VEGF) production and tumour vascularisation in hormone resistant prostate and breast cancer models. Immunofluorescent staining of tumour sections from human oestrogen receptor (ER)-negative breast cancer patients showed a strong correlation between phosphorylated P70S6 kinase (mTOR downstream target), VEGF and SK1 protein expression. In hormone-insensitive prostate (PC3) and breast (MDA-MB-231 and BT-549) cancer cell lines the mTOR inhibitor RAD001 (everolimus) has significantly inhibited SK1 and VEGF expression, while low dose (5 nM) docetaxel had no significant effect. In these cell lines, SK1 overexpression slightly increased the basal levels of VEGF, but did not block the inhibitory effect of RAD001 on VEGF. In a human prostate xenograft model established in nude mice, RAD001 alone or in combination with docetaxel has suppressed tumour growth, VEGF expression and decreased tumour vasculature. Overall, our data demonstrate a new mechanism of an independent regulation of SK1 and VEGF by mTOR in hormone-insensitive prostate and breast cancers.

To increase the removal of middle-sized uremic toxins a new membrane with enhanced permeability and selectivity, called Medium Cut-Off membrane (MCO-Ci) has been developed that at the same time ensures the retention of albumin. Because many middle-sized substances may contribute to micro-inflammation we hypothesized that the use of MCO-Ci influences the inflammatory state in hemodialysis patients.The randomized crossover trial in 48 patients compared MCO-Ci dialysis to High-flux dialysis of 4 weeks duration each plus 8 weeks extension phase. Primary endpoint was the gene expression of TNF-α and IL-6 in peripheral blood mononuclear cells (PBMCs), secondary endpoints were plasma levels of specified inflammatory mediators and cytokines.After four weeks of MCO-Ci the expression of TNF-α mRNA (Relative quantification (RQ) from 0.92 ± 0.34 to 0.75 ± 0.31, -18.5%, p<0.001)-α and IL-6 mRNA (RQ from 0.78 ± 0.80 to 0.60 ± 0.43, -23.1%, p<0.01) was reduced to a significantly greater extent than with High-flux dialyzers (TNF mRNA-RQ: -14.3%; IL-6 mRNA-RQ: -3.5%). After retransformation of logarithmically transformed data, measurements after MCO were reduced to 82% of those after HF (95% CI 74%-91%). 4 weeks use of MCO-Ci resulted in long-lasting change in plasma levels of several cytokines and other substances with a significant decrease for sTNFR1, kappa and lambda free light chains, urea and an increase for Lp-PLA2 (PLA2G7) compared to High-flux. Albumin levels dropped significantly after 4 weeks of MCO dialysis but increased after additional 8 weeks of MCO dialysis. Twelve weeks treatment with MCO-Ci was well tolerated regarding the number of (S)AEs. In the extension period levels of CRP, TNF-α-mRNA and IL-6 mRNA remained stable in High-flux as well as in MCO-Ci.MCO-Ci dialyzers modulate inflammation in chronic HD patients to a greater extent compared to High-flux dialyzers. Transcription of pro-inflammatory cytokines in peripheral leukocytes is markedly reduced and removal of soluble mediators is enhanced with MCO dialysis. Serum albumin concentrations stabilize after an initial drop. These results encourage further trials with longer treatment periods and clinical endpoints.

Many prostate cancers relapse after initial chemotherapy treatment. Combining molecular and chemotherapy together with encapsulation of drugs in nanocarriers provides effective drug delivery and toxicity reduction. We developed core shell lipid-polymer hybrid nanoparticles (CSLPHNPs) with poly (lactic-co-glycolic acid) (PLGA) core and lipid layer containing docetaxel and clinically used inhibitor of sphingosine kinase 1 (SK1) FTY720 (fingolimod). We show for the first time that FTY720 (both free and in CSLPHNPs) re-sensitizes castrate resistant prostate cancer cells and tumors to docetaxel, allowing a four-fold reduction in effective dose. Our CSLPHNPs showed high serum stability and a long shelf life. CSLPHNPs demonstrated a steady uptake by tumor cells, sustained intracellular drug release and in vitro efficacy superior to free therapies. In a mouse model of human prostate cancer, CSLPHNPs showed excellent tumor targeting and significantly lower side effects compared to free drugs, importantly, reversing lymphopenia induced by FTY720. Overall, we demonstrate that nanoparticle encapsulation can improve targeting, provide low off-target toxicity and most importantly reduce FTY720-induced lymphopenia, suggesting its potential use in clinical cancer treatment.

Resistance to docetaxel is a key problem in current prostate cancer management. Sphingosine kinase 1 (SK1) and phosphoinositide 3-kinase (PI3K)/Akt/mammalian target of rapamycin (mTOR) pathways have been implicated in prostate cancer chemoresistance. Here we investigated whether their combined targeting may re-sensitize prostate cancer cells to docetaxel.In hormone-insensitive PC-3 and DU145 prostate cancer cells the mTOR inhibitor everolimus (RAD001) alone did not lead to significant cell death, however, it strongly sensitized cells to low levels (5 nM) of docetaxel. We show that mTOR inhibition has led to a decrease in hypoxia-inducible factor-1α (HIF-1α) protein levels and SK1 mRNA. HIF-1α accumulation induced by CoCl2 has led to a partial chemoresistance to RAD001/docetaxel combination. SK1 overexpression has completely protected prostate cancer cells from RAD001/docetaxel effects. Using gene knockdown and CoCl2 treatment we showed that SK1 mRNA expression is downstream of HIF-1α. In a human xenograft model in nude mice single RAD001 and docetaxel therapies induced 23% and 15% reduction in prostate tumor volume, respectively, while their combination led to a 58% reduction. RAD001 alone or in combination with docetaxel has suppressed intratumoral mTOR and SK1 signaling, however as evidenced by tumor size, it required docetaxel for clinical efficacy. Combination therapy was well tolerated and had similar levels of toxicity to docetaxel alone.Overall, our data demonstrate a new mechanism of docetaxel sensitization in prostate cancer. This provides a mechanistic basis for further clinical application of RAD001/docetaxel combination in prostate cancer therapy.

INTRODUCTION:Patients with chronic kidney disease maintained on intermittent hemodialysis suffer from systemic chronic inflammation which is causally associated with high mortality. Inflammation mediators of 15-45 kDa range cannot be effectively removed by conventional dialysis membranes. In this study, we tested the influence of serum and dialysates obtained from patients maintained on High cut-off or High flux membranes on the inflammation profile of THP-1 monocytes.METHODS:THP-1 monocytes were treated with serum or dialysates obtained from patients maintained on High cut-off and High flux membranes within a randomized crossover pilot trial. Serum-treated cells were subjected to qPCR analyses with TaqMan probes specific for IL6, TNFa, osteopontin and osteocalcin, and transcriptional screening with Inflammatory Array. Apoptosis assay was performed flow cytometrically with 7-AAD and Annexin V staining.FINDINGS:Treatment of the cells with High cut-off serum led to significant reduction of TNFa and IL-6 expression as well as inflammation-related osteopontin and osteocalcin as compared to High flux membrane treatment. As a complementary finding, treatment with High cut-off dialysates induced a pro-apoptotic phenotype in the cells as demonstrated by a significantly increased percentage of 7-AAD and Annexin V positivity. Global screening of serum-treated cells revealed noticeably decreased inflammation profile under High cut-off serum as compared to High flux treatment.DISCUSSION:Taken together, these data demonstrate that High cut-off -membranes eliminate a spectrum of mediators from serum into the dialysate that possess proinflammatory properties and may impair cellular viability.

Sphingosine kinase 1 (SK1) is a lipid enzyme with oncogenic properties that converts the proapoptotic lipids ceramide and sphingosine into the antiapoptotic lipid sphingosine-1-phosphate and activates the signal transduction pathways that lead to cell proliferation, migration, the activation of the inflammatory response, and the impairment of apoptosis.There is compelling evidence that SK1 activation contributes to cancer progression leading to increased oncogenic transformation, tumor growth, resistance to therapies, tumor neovascularization, and metastatic spread. High levels of SK1 expression or activity have been associated with a poor prognosis in several human cancers.Recent studies using cancer cell and mouse models demonstrate a significant potential for SK1-targeting therapies to synergize with the effects of chemotherapy and radiotherapy; however, until recently the absence of clinically applicable SK1 inhibitors has limited the translation of these findings into patients. With the recent discovery of SK1 inhibiting properties of a clinically approved drug FTY720 (Fingolimod), SK1 has gained significant attention from both clinicians and the pharmaceutical industry and it is hoped that trials of newly developed SK1 inhibitors may follow soon.This review provides an overview of the SK1 signaling, its relevance to cancer progression, and the potential clinical significance of targeting SK1 for improved local or systemic control of human cancers.

Chronic inflammation in hemodialysis (HD) patients is associated with cardiovascular complications and mortality. Circulating immune active proteins in the molecular range 15-45 kD that cannot be efficiently cleared by high-flux (HF) dialysis may be causally involved. We intended to test the feasibility of using a high cutoff (HCO) dialyzer in chronic HD patients and its influence on inflammation and monocyte activation. The Gambro HCO1100 dialyzer was compared to a conventional HF membrane in a randomized double-blind crossover trial in 19 chronic HD patients selected for the presence of elevated serum C-reactive protein levels. Patients were treated for six consecutive dialysis sessions (2 weeks) with each membrane. Safety analysis recorded adverse events and albumin losses through the protein-leaking membranes. Efficacy analysis observed reductions in the number of proinflammatory (CD14+CD16+) monocyte subpopulations in circulating blood. Treatment with the HCO membrane was well tolerated, although the number of adverse events was slightly higher. Despite significant serum albumin loss (from 34.1 ± 2.7 to 29.6 ± 3.0 g/L; P < 0.01), there was no need to supplement albumin, and rising activity of cholinesterase during HCO treatment indicated compensation by enhanced hepatic synthesis. The HCO membrane cleared high amounts of proinflammatory cytokines, but did not reduce predialysis inflammatory monocytes and markers. Although the time of HD session was extended, the study was hampered by a lower Kt/V in the HCO compared to the HF period. Treatment of chronic HD patients with this HCO dialyzer for 2 weeks is tolerable in terms of albumin loss and able to clear proinflammatory cytokines; however, this was not sufficient to decrease monocyte activation. Therefore, a more selective, less albumin-leaking membrane is desirable to allow prolonged high-efficient dialysis with more effective cytokine clearance.

Sphingosine kinase 1 (SK1) is a lipid enzyme with oncogenic properties that converts the proapoptotic lipid sphingosine into the antiapoptotic lipid sphingosine-1-phosphate, which activates the signal transduction pathways that lead to cell proliferation, migration, activation of the inflammatory response and impairment of apoptosis. Compelling evidence suggests that SK1 activation contributes to cancer progression leading to increased oncogenic transformation, tumor growth, resistance to therapies, tumor neovascularization and metastatic spread. High levels of SK1 expression or activity have been associated with poor prognosis in several cancers, including those of the prostate. Recent studies using prostate cancer cell and mouse models demonstrate a significant potential for SK1-targeting therapies to synergize with the effects of docetaxel chemotherapy and radiotherapy. However, until recently the absence of clinically applicable SK1 inhibitors has limited the translation of these findings into patients. With the recent discovery that clinically approved drug fingolimod has SK1-inhibiting properties, SK1 has gained significant attention from both clinicians and the pharmaceutical industry and it is hoped that trials of newly developed SK1 inhibitors might follow soon.

Today, most patients who require renal replacement therapy are conventionally treated using dialysis membranes. New developments have been made in the last decade to improve the outcomes for chronic dialysis patients. Cellulose membranes have been replaced by synthetic polymeric membranes with improved biocompatibility, and the development of high-flux membranes and more efficient treatment modes such as hemodiafiltration (HDF) has resulted in improved removal rates for uremic toxins. In addition to membranes used for conventional hemodialysis (HD), new membranes with increased pore size have been developed for specific treatments. These specialized membranes allow the removal of higher-molecular-weight molecules, such as mediators of sepsis/inflammation, or the removal of nephrotoxic immunoglobulin light chains derived from multiple myelomas (bone marrow cancer). However, these membranes permit the passage of all plasma proteins including, for example, albumin. The newest generation of highly selective and permeable medium cut-off (MCO) membranes features the removal of large molecules captured by a high cut-off membrane while simultaneously maintaining low loss of albumin (high-flux membrane). For uremic solutes in the 15,000–45,000 Da size range, MCO membranes offer improved clearance in comparison to high-flux membranes used in HD mode and equivalent clearance to high-flux membranes in high-volume HDF mode. Membranes have also been used in cell-based extracorporeal systems to treat patients with kidney disease. In addition, specialized, highly efficient membranes have been developed to permit stem cell growth in hollow fiber bioreactors as a technological prerequisite for cellular therapies and other applications. Efforts are ongoing to develop wearable or implantable bioartificial kidneys for both chronic and acute kidney failure patients, which represent long-term treatment goals.

Therapeutic drug monitoring is a well-established approach in transplantation medicine to guide immunosuppressive therapy. However, it cannot always predict the effects of immunosuppressive drugs on immune cells, because it does not reflect any aspect of an individual patient's immune system. Pharmacodynamic monitoring is a more recent strategy to provide information about the biologic effect of a specific drug or drug combination on the individual transplant patient. Currently, there is a large number of different biomarkers that either directly (specific markers) or indirectly (global markers) relate to the pharmacodynamic effects of immunosuppressive drugs and are under investigation as potential candidates to be introduced in clinical practice. Such biomarkers may be useful to identify patients at risk of developing acute rejection, infection, or cancer as well as patients who are suitable for minimization of immunosuppressant therapy and may be helpful to manage the timing and rate of immunosuppressant weaning. Serial longitudinal monitoring may allow maintenance of an individualized immunosuppressive regimen. Thus, biomarker monitoring is a potential complementary tool to therapeutic drug monitoring. This review summarizes the current state of knowledge about the use of a number of global or drug-specific pharmacodynamic biomarkers. It is not a comprehensive overview of the literature available, but rather an evidence-based reflection by experts who are intensively involved in scientific work in this field.

Abstract Radiotherapy is widely used as a radical treatment for prostate cancer, but curative treatments are elusive for poorly differentiated tumors where survival is just 15% at 15 years. Dose escalation improves local response rates but is limited by tolerance in normal tissues. A sphingosine analogue, FTY720 (fingolimod), a drug currently in phase III studies for treatment of multiple sclerosis, has been found to be a potent apoptosis inducer in prostate cancer cells. Using in vitro and in vivo approaches, we analyzed the impact of FTY720 on sphingolipid metabolism in hormone-refractory metastatic prostate cancer cells and evaluated its potential as a radiosensitizer on cell lines and prostate tumor xenografts. In prostate cancer cell lines, FTY720 acted as a sphingosine kinase 1 (SphK1) inhibitor that induced prostate cancer cell apoptosis in a manner independent of sphingosine-1-phosphate receptors. In contrast, γ irradiation did not affect SphK1 activity in prostate cancer cells yet synergized with FTY720 to inhibit SphK1. In mice bearing orthotopic or s.c. prostate cancer tumors, we show that FTY720 dramatically increased radiotherapeutic sensitivity, reducing tumor growth and metastasis without toxic side effects. Our findings suggest that low, well-tolerated doses of FTY720 could offer significant improvement to the clinical treatment of prostate cancer. Cancer Res; 70(21); 8651–61. ©2010 AACR.

BACKGROUND AND OBJECTIVES:The effect of mycophenolate mofetil (MMF) on T cell function has not been evaluated in patients undergoing kidney transplantation. The aim of this study was to assess the effect of 1g of MMF on T cell function, that is, intralymphocyte cytokine expression, T cell activation (CD25 and CD71), and T cell proliferation, as well as inosine monophosphate dehydrogenase (IMPDH) activity, to better understand the relationship between pharmacokinetic and pharmacodynamic markers in patients receiving the first dose of MMF before kidney transplantation. PATIENTS:Twenty-four patients undergoing a kidney transplantation from a living donor were enrolled in this study. RESULTS:Compared with baseline (before MMF intake), T cell proliferation (93%), IMPDH activity (74%), CD25 (46%), and CD71 (50%) expression significantly decreased during the first hour after MMF intake, in parallel to the rise in MPA concentration. Thereafter, all pharmacodynamic markers, except IMPDH activity, returned back to baseline level. There was a complex inverse relationship between pharmacokinetic and pharmacodynamic markers. The inhibition of T cell proliferation was highly correlated to IMPDH activity, but also to T cell activation markers. CONCLUSION:The administration of MMF to patients is associated not only with a dramatic decrease in both T cell proliferation and IMPDH activity, but also with in a decrease in CD25 and CD71 expression.

BACKGROUND:In kidney-transplant recipients, leflunomide has been shown to be efficient for treating polyomavirus BK virus-associated-nephropathy (PVAN). However, it is unknown whether the beneficial effect of leflunomide is related to it having a lower immunosuppressive effect than mycophenolate mofetil (MMF), or to its anti-viral activity. The aim of this study was to assess i) T-cell functions before and after conversion from MMF to leflunomide in kidney-transplant patients with PVAN, and ii) effects of leflunomide on PVAN outcome. PATIENTS AND METHODS:Twelve patients were enrolled in this study. At PVAN diagnosis, MMF was replaced by leflunomide. Other immunosuppressive drug doses and levels were maintained unchanged. T-cell functions, i.e., intralymphocyte cytokine expression (IL-2 and TNF-alpha), T-cell activation [i.e., transferrin receptor (CD71) and interleukin (IL)-2 alpha-chain (CD25) expression], and T-cell proliferation were measured using a flow-cytometry whole-blood assay before and at one month after conversion. RESULTS:Despite a slight decrease in tacrolimus trough levels, no significant change in T-cell-function biomarkers was observed after conversion. After a follow-up of 6 (4-30) months, five patients were cleared of the virus, and decreased viral load was observed in four patients. Only one patient suffered a graft loss. No difference in immunological parameters was observed between patients who were cleared or not of BKV. CONCLUSION:Results of this pilot study suggest that the potential benefits of leflunomide to treat PVAN in kidney-transplant patients is not related to reduced immunosuppression induced by replacing MMF by leflunomide. Virological studies are required to determine the anti-BKV effect of leflunomide.

T-cell functions are currently used as biomarkers for the pharmacodynamic monitoring of immunosuppressive drugs or as disease biomarkers of inflammation/sepsis and organ rejection. In order to evaluate co-factors potentially influencing the expression of the immunological biomarkers, we explored T-cell proliferation, T-cell activation (CD25 and CD71 expressions) and intra-lymphocyte cytokine production (interleukin (IL)-2 and tumor necrosis factor (TNF)-alpha) in healthy volunteers, dialysis patients and stable kidney-transplant patients treated with standard immunosuppressive therapy, i.e. tacrolimus, mycophenolic acid with or without steroids. Age was positively correlated with TNF-alpha expression in all three patient populations, and with IL-2 expression in healthy volunteers and kidney-transplant patients. Further age was correlated with inhibition of lymphocyte proliferation in healthy volunteers and with the T-cell activation marker CD25 in kidney-transplant patients. In healthy volunteers lymphocyte proliferation was higher in woman as compared to men. Other biomarkers of T-cell function were independent of the gender. In the kidney-transplant patient group a significantly lower expression of all biomarkers of T-cell functions compared to healthy volunteers and dialysis patients. In dialysis patients we found significant increased IL-2 expression compared to healthy volunteers, while the other T-cell functions were not significantly different. Further time on dialysis had no effect on the level of biomarker expression. In conclusion we found decreased T-cell functions in kidney-transplant patients compared to healthy volunteers and dialysis patients, increased IL-2 expression in dialysis patients compared to healthy volunteers and in all three populations we found a correlation of age and intra-T-lymphocyte TNF-alpha expression.