Adjuvant Radiation after Primary Resection of Atypical Lipomatous Tumors of the Extremity Reduces Local Recurrence but Increases Complications: A Multicenter Evaluation.

Background: Radiation after resection of an atypical lipomatous tumor (ALT) is controversial. This study evaluates local control and complications after the first resection of ALTs of the extremity with or without adjuvant radiation. Methods: A dual institution, retrospective review of patients treated from 1995 to 2020 with first-time resection of an ALT in the extremity was performed. In total, 102 patients underwent adjuvant radiation (XRT group) and 68 patients were treated with surgery alone (no-XRT group). The median follow-up time was 4.6 years (interquartile range (IQR) 2.0-7.3 years). The median radiation dose was 60 Gy (IQR 55-66 Gy). Univariable and multivariable analyses evaluated the association of patient, tumor, and treatment variables with recurrence and complications. Kaplan-Meier analysis evaluated local recurrence-free survival (LRFS) and time to complication. Results: The overall incidence of local recurrence was 1% (1/102) in the XRT group and 24% (16/68) in the no-XRT group (p < 0.001). The median time-to-recurrence was 8.2 years (IQR 6.5-10.5 years). In the XRT and the no-XRT groups, 5-yr LRFS was 98% and 92% (p=0.21) and 10-yr LRFS was 98% and 41% (p < 0.001), respectively. The absence of radiation (HR = 23.63, 95% CI (3.09-180.48); p < 0.001) and R2 surgical resection margins (HR = 11.04, 95% CI (2.07-59.03); p < 0.001) incurred a 23-fold and 11-fold increased risk of local recurrence, respectively, while tumor size, depth, location, and neurovascular involvement were not found to be independent predictors of recurrence. The complication rate was 37% (38/102) in the XRT group and 10% (7/68) in the no-XRT group (p < 0.001). Eight patients (8/102, 8%) required surgical management for complication in the XRT group compared with two patients (2/68, 3%) in the no-XRT group (p=0.10). Higher radiation dose had a modest correlation with increased severity of complication (ρ=0.24; p=0.02). Conclusions: Adjuvant radiation after first-time resection of an ALT of the extremity was associated with a significantly reduced risk of local recurrence but a three-fold increase in complication rate. These data support a 10-year follow-up for these patients and inform a notable clinical trade-off if considering adjuvant radiation for this tumor with recurrent potential.

Multicenter, retrospective comparison of implant survival, complications and cost between plate and screw and intramedullary nail fixation for metastatic lesions of the diaphyseal humerus.

Background: The humerus is a common site of metastatic disease that can be fixated with either plate and screw or intramedullary nail (IMN) constructs. A multicenter retrospective comparison study was undertaken to compare implant survival, complication rate and cost between the two constructs. No prior studies have included a cost comparison. Methods: Databases of two academic practices were queried retrospectively to identify patients with metastases of the humerus. Inclusion criteria were a lesion in the proximal metaphysis to distal diaphysis and amenable to both implant options with available cost data. Follow-up was at least 6 months barring death or discharge to hospice sooner. Demographic, clinical and outcome data was recorded. Costs were estimated based on contract pricing. Operating room (OR) costs were estimated using per minute OR costs proposed by other investigators. Results: One hundred and one humeri in 96 patients were included (72 plates and 29 nails). The most common malignancies were renal cell, myeloma and lung. Half presented with a displaced fracture. Demographics were similar in both groups. Lesions were larger in the plate group. Surgical times were longer in the plate group, 146 vs. 75 min, P<0.001. Estimated blood loss (EBL) was higher in the plate group, 510 vs. 221 mL, P<0.001. A trend toward increased failure was seen in the plate group, 12.5% vs. 0% (P=0.056). The most common complications in the plate group were pain, stiffness and swelling compared to pain, refracture and PE in the nail group. Local disease progression was equivalent. Implant costs were higher in the IMN group ($2,753 vs. $1,553, P<0.001), while OR costs were lower ($2,349 vs. $4,395, P<0.001). Overall cost of implantation was lower in the IMN group ($5,102 vs. $5,949, P=0.005). Conclusions: IMN of metastases of the humerus offers a faster, potentially more durable construct with lower blood loss, faster OR times and decreased cost of implantation.

Ceramide Synthase 6 Maximizes p53 Function to Prevent Progeny Formation from Polyploid Giant Cancer Cells.

Polyploid giant cancer cells (PGCC) constitute a transiently senescent subpopulation of cancer cells that arises in response to stress. PGCC are capable of generating progeny via a primitive, cleavage-like cell division that is dependent on the sphingolipid enzyme acid ceramidase (ASAH1). The goal of this study was to understand differences in sphingolipid metabolism between non-polyploid and polyploid cancer cells to gain an understanding of the ASAH1-dependence in the PGCC population. Steady-state and flux analysis of sphingolipids did not support our initial hypothesis that the ASAH1 product sphingosine is rapidly converted into the pro-survival lipid sphingosine-1-phosphate. Instead, our results suggest that ASAH1 activity is important for preventing the accumulation of long chain ceramides such as C16-ceramide. We therefore determined how modulation of C16-ceramide, either through CerS6 or p53, a known PGCC suppressor and enhancer of CerS6-derived C16-ceramide, affected PGCC progeny formation. Co-expression of the CerS6 and p53 abrogated the ability of PGCC to form offspring, suggesting that the two genes form a positive feedback loop. CerS6 enhanced the effect of p53 by significantly increasing protein half-life. Our results support the idea that sphingolipid metabolism is of functional importance in PGCC and that targeting this signaling pathway has potential for clinical intervention.

Inhibition of acid ceramidase regulates MHC class II antigen presentation and suppression of autoimmune arthritis.

The bioactive sphingolipid ceramide affects immune responses although its effect on antigen (Ag) processing and delivery by HLA class II to CD4+T-cells remains unclear. Therefore, we examined the actions of a novel cell-permeable acid ceramidase (AC) inhibitor [(1R,2R) N myristoylamino-(4'-nitrophenyl)-propandiol-1,3] on antigen presentation and inflammatory cytokine production by Ag-presenting cells (APCs) such as B-cells, macrophages, and dendritic cells. We found that AC inhibition in APCs perturbed Ag-processing and presentation via HLA-DR4 (MHC class II) proteins as measured by coculture assay and T-cell production of IL-2. Mass spectral analyses showed that B13 treatment significantly raised levels of four types of ceramides in human B-cells. B13 treatment did not alter Ag internalization and class II protein expression, but significantly inhibited lysosomal cysteinyl cathepsins (B, S and L) and thiol-reductase (GILT), HLA class II Ag-processing, and generation of functional class II-peptide complexes. Ex vivo Ag presentation assays showed that inhibition of AC impaired primary and recall CD4+T-cell responses and cytokine production in response against type II collagen. Further, B13 delayed onset and reduced severity of inflamed joints and cytokine production in the collagen-induced arthritis mouse model in vivo. These findings suggest that inhibition of AC in APCs may dysregulate endolysosomal proteases and HLA class II-associated self-antigen presentation to CD4+T-cells, attenuating inflammatory cytokine production and suppressing host autoimmune responses.

Noninvasive diffusion magnetic resonance imaging of brain tumour cell size for the early detection of therapeutic response.

Cancer cells differ in size from those of their host tissue and are known to change in size during the processes of cell death. A noninvasive method for monitoring cell size would be highly advantageous as a potential biomarker of malignancy and early therapeutic response. This need is particularly acute in brain tumours where biopsy is a highly invasive procedure. Here, diffusion MRI data were acquired in a GL261 glioma mouse model before and during treatment with Temozolomide. The biophysical model VERDICT (Vascular Extracellular and Restricted Diffusion for Cytometry in Tumours) was applied to the MRI data to quantify multi-compartmental parameters connected to the underlying tissue microstructure, which could potentially be useful clinical biomarkers. These parameters were compared to ADC and kurtosis diffusion models, and, measures from histology and optical projection tomography. MRI data was also acquired in patients to assess the feasibility of applying VERDICT in a range of different glioma subtypes. In the GL261 gliomas, cellular changes were detected according to the VERDICT model in advance of gross tumour volume changes as well as ADC and kurtosis models. VERDICT parameters in glioblastoma patients were most consistent with the GL261 mouse model, whilst displaying additional regions of localised tissue heterogeneity. The present VERDICT model was less appropriate for modelling more diffuse astrocytomas and oligodendrogliomas, but could be tuned to improve the representation of these tumour types. Biophysical modelling of the diffusion MRI signal permits monitoring of brain tumours without invasive intervention. VERDICT responds to microstructural changes induced by chemotherapy, is feasible within clinical scan times and could provide useful biomarkers of treatment response.

Tamoxifen is a candidate first-in-class inhibitor of acid ceramidase that reduces amitotic division in polyploid giant cancer cells-Unrecognized players in tumorigenesis.

Polyploid giant cancer cells (PGCC) represent a poorly understood, small subpopulation of tumor cells that are increasingly being recognized for their critical role in therapy resistance, metastasis, and cancer recurrence. PGCC have the potential to generate progeny through primitive or cleavage-like division, which allows them to evade antimitotic insults. We recently demonstrated that the sphingolipid enzyme acid ceramidase (ASAH1) is required for this process. Since specific ASAH1 inhibitors are not clinically available, we investigated whether tamoxifen, which interferes with ASAH1 function via off-target effects, has a potential clinical benefit independent of estrogen signaling. Our results show that tamoxifen inhibits generation of PGCC offspring in prostate cancer, glioblastoma, and melanoma cells. Analysis of two state-level cancer registries revealed that tamoxifen improves survival outcomes for second, nonbreast cancers that develop in women with early stage breast cancer. Our results suggest that tamoxifen may have a clinical benefit in a variety of cancers that is independent of estrogen signaling and could be due to its inhibition of acid ceramidase. Thus the distinct application of tamoxifen as potentially a first-in-class therapeutic that inhibits the generation of PGCC offspring should be considered in future clinical trials.

PROTAC-mediated degradation of class I histone deacetylase enzymes in corepressor complexes.

We have identified a proteolysis targeting chimera (PROTAC) of class I HDACs 1, 2 and 3. The most active degrader consists of a benzamide HDAC inhibitor, an alkyl linker, and the von Hippel-Lindau E3 ligand. Our PROTAC increased histone acetylation levels and compromised colon cancer HCT116 cell viability, establishing a degradation strategy as an alternative to class I HDAC inhibition.

Measuring diffusion exchange across the cell membrane with DEXSY (Diffusion Exchange Spectroscopy).

INTRODUCTION: To combine numerical simulations, in vitro and in vivo experiments to evaluate the feasibility of measuring diffusion exchange across the cell membrane with diffusion exchange spectroscopy (DEXSY). METHODS: DEXSY acquisitions were simulated over a range of permeabilities in nerve tissue and yeast substrates. In vitro measurements were performed in a yeast substrate and in vivo measurements in mouse tumor xenograft models, all at 9.4 T. RESULTS: Diffusion exchange was observed in simulations over a physiologically relevant range of cell permeability values. In vitro and in vivo measures also provided evidence of diffusion exchange, which was quantified with the Diffusion Exchange Index (DEI). CONCLUSIONS: Our findings provide preliminary evidence that DEXSY can be used to make in vivo measurements of diffusion exchange and cell membrane permeability.

Inadvertent Inguinal Sarcoma Excision during Hernia Surgery: Outcomes, Gender Analysis, and Prevention.

INTRODUCTION: Inadvertent excision of a soft tissue sarcoma during hernia surgery is a preventable clinical scenario that leads to unnecessary patient morbidity. Prior series are few, which only include male patients with little focus on prevention. The purpose of this study is to report the presenting features and outcomes of both male and female patients who underwent inadvertent inguinal sarcoma excision during hernia surgery. METHODS: A retrospective analysis of a single sarcoma referral center identified 33 patients who were referred for definitive treatment. Patients were divided into three clinically relevant groups based on intraoperative diagnosis, sex, and location of the mass relative to the inguinal ligament. T-tests and Fisher's exact tests were performed to compare continuous and categorical variables, respectively. Kaplan-Meier modeling was performed to assess sarcoma-specific survival. RESULTS: Females were younger (47 years vs. 61 years, p=0.003) and had smaller sarcomas (6.7 cm vs. 11 cm, p=0.012) compared to males. Only two sarcomas (2/33, 6%) were <4 cm in size. The majority of sarcomas in females were above the inguinal ligament (12/14, 86%). Twenty-nine (88%) underwent definitive R0 excision. The mean number of surgeries per patient was three (range 1-13), with nineteen (58%) patients requiring flap reconstruction and six (18%) requiring vascular bypass. Five patients locally recurred (15%) at a mean of 38 months after definitive excision (range 5-128 months). Overall sarcoma-specific disease-free survival was 64%, with no difference between males (80 ± 11%) and females (59 ± 17%) (p=0.885). Mean follow-up was 75 months (range 5-212). CONCLUSION: This is the second largest study regarding inadvertent inguinal sarcoma excision and the first to include females. When a suspected hernia is >4 cm, irreducible, firm, and is growing, especially in females, consider obtaining preoperative advanced three-dimensional imaging (CT or MRI) that can differentiate a neoplasm from a hernia.

Siderophore-Antibiotic Conjugate Design: New Drugs for Bad Bugs?

Antibiotic resistance is a global health concern and a current threat to modern medicine and society. New strategies for antibiotic drug design and delivery offer a glimmer of hope in a currently limited pipeline of new antibiotics. One strategy involves conjugating iron-chelating microbial siderophores to an antibiotic or antimicrobial agent to enhance uptake and antibacterial potency. Cefiderocol (S-649266) is a promising cephalosporin-catechol conjugate currently in phase III clinical trials that utilizes iron-mediated active transport and demonstrates enhanced potency against multi-drug resistant (MDR) Gram-negative pathogens. Such molecules demonstrate that siderophore-antibiotic conjugates could be important future medicines to add to our antibiotic arsenal. This review is written in the context of the chemical design of siderophore-antibiotic conjugates focusing on the differing siderophore, linker, and antibiotic components that make up conjugates. We selected chemically distinct siderophore-antibiotic conjugates as exemplary conjugates, rather than multiple analogues, to highlight findings to date. The review should offer a general guide to the uninitiated in the molecular design of siderophore-antibiotic conjugates.

Genetic and pharmacological inhibition of acid ceramidase prevents asymmetric cell division by neosis.

Radiation treatment failure or relapse after initial response to chemotherapy presents significant clinical challenges in cancer patients. Escape from initial courses of treatment can involve reactivation of embryonic developmental stages, with the formation of polynuclear giant cancer cells (PGCCs). This strategy of dedifferentiation can insulate cancer cells from a variety of treatments and allows a residual subpopulation to reestablish tumors after treatment. Using radiation or docetaxel chemotherapy, we generated PGCCs from prostate cancer cells. Here, we show that expression of acid ceramidase (ASAH1), an enzyme in the sphingolipid pathway linked to therapy resistance and poor outcomes, is elevated in PGCCs. Targeting ASAH1 with shRNA or treatment with the ASAH1 inhibitor, LCL-521, did not impair the formation of PGCCs, but prevented the formation of PGCC progeny that arise through an asymmetric cell division called neosis. Similar results were obtained in lung cancer cells that had been exposed to radiation or cisplatin chemotherapy as stressors. In summary, our data suggest that endoreplication occurs independent of ASAH1 while neosis is ASAH1-dependent in both prostate and lung cancer cells. Because ASAH1 knockout is embryonic lethal but not deleterious to adult animals, targeting this enzyme has the potential to be highly specific to cells undergoing the dedifferentiation process to escape cancer treatments. Pharmacological inhibition of ASAH1 is a potentially powerful strategy to eliminate cells that could otherwise serve as seed populations for recurrence.

Talk CPR - a technology project to improve communication in do not attempt cardiopulmonary resuscitation decisions in palliative illness.

BACKGROUND: A national Do Not Attempt Cardiopulmonary Resuscitation policy was rolled out for the National Health Service in Wales in 2015. A national steering group led on producing information videos and a website for patients, carers and healthcare professionals, forming part of a quality improvement program. Videos were planned, scripted and produced with healthcare professionals and patient/carer representatives, and were completed with both English and Welsh language versions. The TalkCPR videos encourage and promote open discussion about Cardiopulmonary Resuscitation (CPR) and DNACPR in palliative care situations. METHODS: We worked with patient/carer groups to evaluate whether video resources to convey the salient facts involved in CPR and DNACPR decisions for people with palliative and life-limiting illness were acceptable or not. We conducted a mixed-method design service review in five phases to evaluate whether this technological resource could help. After creating video and website materials, they were evaluated by doctors, nurses and a patient/carer group. We also sent out one lightweight TalkCPR video media pad to each practice in Wales. These rechargeable electronic video media pads had communication videos pre-loaded for easy viewing, especially in areas with poor roaming data coverage. RESULTS: Videos were demonstrably acceptable to both patient and carer groups, and improved healthcare professional confidence and understanding. Videos went live on the TalkCPR website, in all Welsh Health Boards and on Youtube, and are now used in routine practice throughout Wales. CONCLUSION: This is the first time that DNACPR information videos are aimed directly at palliative care patients and carers, to explore this sensitive subject with them, and to encourage them to approach their doctor or nurse about it. The website, app and video media pads were developed by patients, the Digital Legacy Association, Welsh NHS IT services, Welsh Government, the Bevan Commission and the Dying Matters Charity in Wales 'Byw Nawr'. The GMC, the Royal College of General Practitioners and NICE have listed TalkCPR as a learning resource. There has also been a collaboration with Falmouth University Art College, who helped produce graphic designs to facilitate and encourage discussions about CPR and end of life care.

Interdiction of Sphingolipid Metabolism Revisited: Focus on Prostate Cancer.

Sphingolipid metabolism is known to play a role in cell death, survival, and therapy resistance in cancer. Sphingolipids, particularly dihydroceramide and ceramide, are associated with antiproliferative or cell death responses, respectively, and are central to effective cancer therapy. Within the last decade, strides have been made in elucidating many intricacies of sphingolipid metabolism. New information has emerged on the mechanisms by which sphingolipid metabolism is dysregulated during malignancy and how cancer cells survive and/or escape therapeutic interventions. This chapter focuses on three main themes: (1) sphingolipid enzymes that are dysregulated in cancer, particularly in prostate cancer; (2) inhibitors of sphingolipid metabolism that antagonize prosurvival responses; and (3) sphingolipid-driven escape mechanisms that allow cancer cells to evade therapies. We explore clinical and preclinical approaches to interdict sphingolipid metabolism and provide a rationale for combining strategies to drive the generation of antiproliferative ceramides with prevention of ceramide clearance.

Blocking Myristoylation of Src Inhibits Its Kinase Activity and Suppresses Prostate Cancer Progression.

Protein N-myristoylation enables localization to membranes and helps maintain protein conformation and function. N-myristoyltransferases (NMT) catalyze co- or posttranslational myristoylation of Src family kinases and other oncogenic proteins, thereby regulating their function. In this study, we provide genetic and pharmacologic evidence that inhibiting the N-myristoyltransferase NMT1 suppresses cell-cycle progression, proliferation, and malignant growth of prostate cancer cells. Loss of myristoylation abolished the tumorigenic potential of Src and its synergy with androgen receptor in mediating tumor invasion. We identified the myristoyl-CoA analogue B13 as a small-molecule inhibitor of NMT1 enzymatic activity. B13 exposure blocked Src myristoylation and Src localization to the cytoplasmic membrane, attenuating Src-mediated oncogenic signaling. B13 exerted its anti-invasive and antitumor effects against prostate cancer cells, with minimal toxic side-effects in vivo Structural optimization based on structure-activity relationships enabled the chemical synthesis of LCL204, with enhanced inhibitory potency against NMT1. Collectively, our results offer a preclinical proof of concept for the use of protein myristoylation inhibitors as a strategy to block prostate cancer progression. Cancer Res; 77(24); 6950-62. ©2017 AACR.

Acid Ceramidase Deficiency is characterized by a unique plasma cytokine and ceramide profile that is altered by therapy.

Acid Ceramidase Deficiency (Farber disease, FD) is an ultra-rare Lysosomal Storage Disorder that is poorly understood and often misdiagnosed as Juvenile Idiopathic Arthritis (JIA). Hallmarks of FD are accumulation of ceramides, widespread macrophage infiltration, splenomegaly, and lymphocytosis. The cytokines involved in this abnormal hematopoietic state are unknown. There are dozens of ceramide species and derivatives, but the specific ones that accumulate in FD have not been investigated. We used a multiplex assay to analyze cytokines and mass spectrometry to analyze ceramides in plasma from patients and mice with FD, controls, Farber patients treated by hematopoietic stem cell transplantation (HSCT), JIA patients, and patients with Gaucher disease. KC, MIP-1α, and MCP-1 were sequentially upregulated in plasma from FD mice. MCP-1, IL-10, IL-6, IL-12, and VEGF levels were elevated in plasma from Farber patients but not in control or JIA patients. C16-Ceramide (C16-Cer) and dhC16-Cer were upregulated in plasma from FD mice. a-OH-C18-Cer, dhC12-Cer, dhC24:1-Cer, and C22:1-Cer-1P accumulated in plasma from patients with FD. Most cytokines and only a-OH-C18-Cer returned to baseline levels in HSCT-treated Farber patients. Sphingosines were not altered. Chitotriosidase activity was also relatively low. A unique cytokine and ceramide profile was seen in the plasma of Farber patients that was not observed in plasma from HSCT-treated Farber patients, JIA patients, or Gaucher patients. The cytokine profile can potentially be used to prevent misdiagnosis of Farber as JIA and to monitor the response to treatment. Further understanding of why these signaling molecules and lipids are elevated can lead to better understanding of the etiology and pathophysiology of FD and inform development of future treatments.

Tissue biomarkers of drug efficacy: case studies using a MALDI-MSI workflow.

MALDI MS imaging (MALDI-MSI) offers a capability to not only evaluate the distribution, localization and metabolism of drugs within tissues but also allow correlative tissue measurement of the effect of the drug on biomolecules in the targeted pathway. Particularly for MALDI-MSI, lipid molecules are readily detectable within tissues. Case study examples are provided for two different drugs targeting the sphingosine-1-phosphate/ceramide nexus in tumor xenograft tissues. A workflow combining high-resolution MALDI-MSI with on-tissue confirmation of targeted compounds using a structural library and on-tissue enzymatic digestion strategy is described. Representative images of drug metabolite distribution that correlate to an increase or decrease in sphingosine-1-phosphate or ceramide species are provided.

Enterobacteria-secreted particles induce production of exosome-like S1P-containing particles by intestinal epithelium to drive Th17-mediated tumorigenesis.

Gut-associated inflammation plays a crucial role in the progression of colon cancer. Here, we identify a novel pathogen-host interaction that promotes gut inflammation and the development of colon cancer. We find that enteropathogenic bacteria-secreted particles (ET-BSPs) stimulate intestinal epithelium to produce IDENs (intestinal mucosa-derived exosome-like nanoparticles) containing elevated levels of sphingosine-1-phosphate, CCL20 and prostaglandin E2 (PGE2). CCL20 and PGE2 are required for the recruitment and proliferation, respectively, of Th17 cells, and these processes also involve the MyD88-mediated pathway. By influencing the recruitment and proliferation of Th17 cells in the intestine, IDENs promote colon cancer. We demonstrate the biological effect of sphingosine-1-phosphate contained in IDENs on tumour growth in spontaneous and transplanted colon cancer mouse models. These findings provide deeper insights into how host-microbe relationships are mediated by particles secreted from both bacterial and host cells.

Sphingosine 1-Phosphate Receptor 2 Regulates the Migration, Proliferation, and Differentiation of Mesenchymal Stem Cells.

Mesenchymal stem cells (MSCs) are a multipotent cell population acquired most prominently from bone marrow with the capacity to differentiate into osteoblasts, chondrocytes, adipocytes, and others. MSCs demonstrate the capacity to home to sites of injury and contribute to tissue repair. Sphingosine 1-phosphate (S1P) is a biologically active sphingolipid impacting proliferation, apoptosis, inflammation, and angiogenesis with changes in S1P concentration providing significant implications for various disease conditions including cancer, diabetes, and cardiac disease. These functions are primarily mediated by interactions with 5 G-protein coupled S1P receptors (S1PR1-5). In this paper, we demonstrate that inhibition of S1PR2 results in increased MSC clonogenicity, migration, and proliferation; features dependent on Erk phosphorylation. Furthermore, decreased S1PR2 expression decreases the differentiation of MSCs into adipocytes and mature osteoblasts that may be the result of increased expression of MSC pluripotency factors including Nanog, Sox-9, and Oct-4. Inhibition of S1PR1 and S1PR3 in contrast does not impact MSC migration or Erk activation although increased proliferation is observed. In the study, we describe the essential role of S1PR2 in MSC differentiation pathways through modification of pluripotency factors. We propose a MAPK dependent mechanism through S1PR2 inhibition that promotes equally multipotent MSC proliferation.

Targeting (cellular) lysosomal acid ceramidase by B13: design, synthesis and evaluation of novel DMG-B13 ester prodrugs.

Acid ceramidase (ACDase) is being recognized as a therapeutic target for cancer. B13 represents a moderate inhibitor of ACDase. The present study concentrates on the lysosomal targeting of B13 via its N,N-dimethylglycine (DMG) esters (DMG-B13 prodrugs). Novel analogs, the isomeric mono-DMG-B13, LCL522 (3-O-DMG-B13·HCl) and LCL596 (1-O-DMG-B13·HCl) and di-DMG-B13, LCL521 (1,3-O, O-DMG-B13·2HCl) conjugates, were designed and synthesized through N,N-dimethyl glycine (DMG) esterification of the hydroxyl groups of B13. In MCF7 cells, DMG-B13 prodrugs were efficiently metabolized to B13. The early inhibitory effect of DMG-B13 prodrugs on cellular ceramidases was ACDase specific by their lysosomal targeting. The corresponding dramatic decrease of cellular Sph (80-97% Control/1h) by DMG-B13 prodrugs was mainly from the inhibition of the lysosomal ACDase.