Mutant IDH regulates glycogen metabolism from early cartilage development to malignant chondrosarcoma formation.

Chondrosarcomas are the most common malignancy of cartilage and are associated with somatic mutations in isocitrate dehydrogenase 1 (IDH1) and IDH2 genes. Somatic IDH mutations are also found in its benign precursor lesion, enchondromas, suggesting that IDH mutations are early events in malignant transformation. Human mutant IDH chondrosarcomas and mutant Idh mice that develop enchondromas investigated in our studies display glycogen deposition exclusively in mutant cells from IDH mutant chondrosarcomas and Idh1 mutant murine growth plates. Pharmacologic blockade of glycogen utilization induces changes in tumor cell behavior, downstream energetic pathways, and tumor burden in vitro and in vivo. Mutant IDH1 interacts with hypoxia-inducible factor 1α (HIF1α) to regulate expression of key enzymes in glycogen metabolism. Here, we show a critical role for glycogen in enchondromas and chondrosarcomas, which is likely mediated through an interaction with mutant IDH1 and HIF1α.

CD142 Identifies Neoplastic Desmoid Tumor Cells, Uncovering Interactions Between Neoplastic and Stromal Cells That Drive Proliferation.

The interaction between neoplastic and stromal cells within a tumor mass plays an important role in cancer biology. However, it is challenging to distinguish between tumor and stromal cells in mesenchymal tumors because lineage-specific cell surface markers typically used in other cancers do not distinguish between the different cell subpopulations. Desmoid tumors consist of mesenchymal fibroblast-like cells driven by mutations stabilizing beta-catenin. Here we aimed to identify surface markers that can distinguish mutant cells from stromal cells to study tumor-stroma interactions. We analyzed colonies derived from single cells from human desmoid tumors using a high-throughput surface antigen screen, to characterize the mutant and nonmutant cells. We found that CD142 is highly expressed by the mutant cell populations and correlates with beta-catenin activity. CD142-based cell sorting isolated the mutant population from heterogeneous samples, including one where no mutation was previously detected by traditional Sanger sequencing. We then studied the secretome of mutant and nonmutant fibroblastic cells. PTX3 is one stroma-derived secreted factor that increases mutant cell proliferation via STAT6 activation. These data demonstrate a sensitive method to quantify and distinguish neoplastic from stromal cells in mesenchymal tumors. It identifies proteins secreted by nonmutant cells that regulate mutant cell proliferation that could be therapeutically. Significance: Distinguishing between neoplastic (tumor) and non-neoplastic (stromal) cells within mesenchymal tumors is particularly challenging, because lineage-specific cell surface markers typically used in other cancers do not differentiate between the different cell subpopulations. Here, we developed a strategy combining clonal expansion with surface proteome profiling to identify markers for quantifying and isolating mutant and nonmutant cell subpopulations in desmoid tumors, and to study their interactions via soluble factors.

Structural basis of broad-spectrum ß-lactam resistance in Staphylococcus aureus.

Broad-spectrum ß-lactam antibiotic resistance in Staphylococcus aureus is a global healthcare burden1,2. In clinical strains, resistance is largely controlled by BlaR13, a receptor that senses ß-lactams through the acylation of its sensor domain, inducing transmembrane signalling and activation of the cytoplasmic-facing metalloprotease domain4. The metalloprotease domain has a role in BlaI derepression, inducing blaZ (ß-lactamase PC1) and mecA (ß-lactam-resistant cell-wall transpeptidase PBP2a) expression3-7. Here, overcoming hurdles in isolation, we show that BlaR1 cleaves BlaI directly, as necessary for inactivation, with no requirement for additional components as suggested previously8. Cryo-electron microscopy structures of BlaR1-the wild type and an autocleavage-deficient F284A mutant, with or without ß-lactam-reveal a domain-swapped dimer that we suggest is critical to the stabilization of the signalling loops within. BlaR1 undergoes spontaneous autocleavage in cis between Ser283 and Phe284 and we describe the catalytic mechanism and specificity underlying the self and BlaI cleavage. The structures suggest that allosteric signalling emanates from ß-lactam-induced exclusion of the prominent extracellular loop bound competitively in the sensor-domain active site, driving subsequent dynamic motions, including a shift in the sensor towards the membrane and accompanying changes in the zinc metalloprotease domain. We propose that this enhances the expulsion of autocleaved products from the active site, shifting the equilibrium to a state that is permissive of efficient BlaI cleavage. Collectively, this study provides a structure of a two-component signalling receptor that mediates action-in this case, antibiotic resistance-through the direct cleavage of a repressor.

18F-FET-PET imaging in high-grade gliomas and brain metastases: a systematic review and meta-analysis.

PURPOSE: To provide a summary of the diagnostic performance of 18F-FET-PET in the management of patients with high-grade brain gliomas or metastases from extracranial primary malignancies. METHODS: MEDLINE, EMBASE, and Cochrane Database of Systematic Reviews databases were searched for studies that reported on diagnostic test parameters in radiotherapy planning, response assessment, and tumour recurrence/treatment-related changes differentiation. Radiomic studies were excluded. Quality assessment was performed using the Quality Assessment of Diagnostic Accuracy Studies (QUADAS-2) tool and the GRADE approach. A bivariate, random-effects model was used to produce summary estimates of sensitivity and specificity. RESULTS: Twenty-six studies with a total of 1206 patients/lesions were included in the analysis. For radiotherapy planning of glioma, the pooled proportion of patients from 3 studies with 18F-FET uptake extending beyond the 20 mm margin from the gadolinium enhancement on standard MRI was 39% (95% CI, 10-73%). In 3 studies, 18F-FET-PET was also shown to be predictive of early responders to treatment, whereas MRI failed to show any prognostic value. For the differentiation of glioma recurrence from treatment-related changes, the pooled sensitivity and specificity of TBRmax 1.9-2.3 from 6 studies were 91% (95% CI, 74-97%) and 84% (95% CI, 69-93%), respectively. The respective values for brain metastases from 4 studies were 82% (95% CI, 74-88%) and 82% (95% CI, 74-88%) using TBRmax 2.15-3.11. CONCLUSION: While 18F-FET shows promise as a complementary modality to standard-of-care MRI for the management of primary and metastatic brain malignancies, further validation with standardized image interpretation methods in well-designed prospective studies are warranted.

In Vivo Detection of Cyclic-di-AMP in Staphylococcus aureus.

Cyclic-di-AMP (CDA) is a signaling molecule that controls various cellular functions including antibiotic tolerance and osmoregulation in Staphylococcus aureus (S. aureus). In this study, we developed a novel biosensor (bsuO P6-4) for in vivo detection of CDA in S. aureus. The fluorescent biosensor is based on a natural CDA riboswitch from Bacillus subtilis connected at its P6 stem to the dye-binding aptamer Spinach. Our study showed that bsuO P6-4 could detect a wide concentration range of CDA in both laboratory and clinical strains, making it suitable for use in both basic and clinical research applications.

Neoadjuvant and Adjuvant Systemic Therapy for Newly Diagnosed Stage II-IV Epithelial Ovary, Fallopian Tube, or Primary Peritoneal Carcinoma: A Practice Guideline.

BACKGROUND: This study aims to provide guidance for the use of neoadjuvant and adjuvant systemic therapy in women with newly diagnosed stage II-IV epithelial ovary, fallopian tube, or primary peritoneal carcinoma. METHODS: EMBASE, MEDLINE, and Cochrane Library were investigated for relevant systematic reviews and phase III trials. Articles focusing on consolidation and maintenance therapies were excluded. RESULTS: For women with potentially resectable disease, primary cytoreductive surgery, followed by six to eight cycles of intravenous three-weekly paclitaxel and carboplatin is recommended. For those with a high-risk profile for primary cytoreductive surgery, neoadjuvant chemotherapy can be an option. Adjuvant chemotherapy with six cycles of dose-dense weekly paclitaxel plus three-weekly carboplatin can be considered for women of Japanese descent. In women with stage III or IV disease, the incorporation of bevacizumab concurrent with paclitaxel and carboplatin is not recommended for use as adjuvant therapy unless bevacizumab is continued as maintenance therapy. Intravenous paclitaxel plus intraperitoneal cisplatin and paclitaxel can be considered for stage III optimally debulked women who did not receive neoadjuvant chemotherapy. However, intraperitoneal administration of chemotherapy with bevacizumab should not be considered as an option for stage II-IV optimally debulked women. DISCUSSION: The recommendations represent a current standard of care that is feasible to implement and valued by both clinicians and patients.

Loss of GdpP Function in Staphylococcus aureus Leads to ß-Lactam Tolerance and Enhanced Evolution of ß-Lactam Resistance.

Infections caused by Staphylococcus aureus are a leading cause of mortality. Treating infections caused by S. aureus is difficult due to resistance against most traditional antibiotics, including ß-lactams. We previously reported the presence of mutations in gdpP among S. aureus strains that were obtained by serial passaging in ß-lactam drugs. Similar mutations have recently been reported in natural S. aureus isolates that are either nonsusceptible or resistant to ß-lactam antibiotics. gdpP codes for a phosphodiesterase that cleaves cyclic-di-AMP (CDA), a newly discovered second messenger. In this study, we sought to identify the role of gdpP in ß-lactam resistance in S. aureus. Our results showed that gdpP-associated mutations caused loss of phosphodiesterase function, leading to increased CDA accumulation in the bacterial cytosol. Deletion of gdpP led to an enhanced ability of the bacteria to withstand a ß-lactam challenge (2 to 3 log increase in bacterial CFU) by promoting tolerance without enhancing MICs of ß-lactam antibiotics. Our results demonstrated that increased drug tolerance due to loss of GdpP function can provide a selective advantage in acquisition of high-level ß-lactam resistance. Loss of GdpP function thus increases tolerance to ß-lactams that can lead to its therapy failure and can permit ß-lactam resistance to occur more readily.

PBP4-mediated ß-lactam resistance among clinical strains of Staphylococcus aureus.

BACKGROUND: PBP4, a low-molecular-weight PBP in Staphylococcus aureus, is not considered to be a classical mediator of ß-lactam resistance. Previous studies carried out by our group with laboratory strains of S. aureus demonstrated the ability of PBP4 to produce ß-lactam resistance through mutations associated with the pbp4 promoter and/or gene. Recent studies of ß-lactam-resistant clinical isolates of S. aureus have reported similar mutations associated with pbp4. OBJECTIVES: To determine if pbp4-associated mutations reported among clinical strains of S. aureus mediate ß-lactam resistance. METHODS: The pbp4 promoters and genes bearing mutations from clinical isolates were cloned into a heterologous host. Reporter, growth and Bocillin assays were performed to assess their role in ß-lactam resistance. X-ray crystallography was used to obtain acyl-enzyme intermediate structures of the WT and mutant PBP4 with nafcillin and cefoxitin. RESULTS: Of the five strains that contained pbp4 promoter mutations, three strains exhibited enhanced expression of PBP4. The R200L mutation in pbp4 resulted in increased survival in the presence of the ß-lactams nafcillin and cefoxitin. Further, introduction of either a promoter or a gene mutation into the genome of a WT host increased the ability of the strains to resist the action of ß-lactams. The four high-resolution X-ray structures presented demonstrate the binding pose of the ß-lactams tested and provide hints for further drug development. CONCLUSIONS: Mutations associated with the pbp4 promoter and pbp4 gene altered protein activity and mediated ß-lactam resistance among the clinically isolated strains that were studied.

Neoadjuvant and adjuvant systemic therapy for newly diagnosed stage II- IV epithelial ovary, fallopian tube, or primary peritoneal carcinoma: A systematic review.

BACKGROUND: To systematically review neoadjuvant and adjuvant therapy options for women with newly diagnosed stage II-IV ovarian cancer. METHODS: Phase III trials were searched using MEDLINE, EMBASE, and Cochrane Library. Maintenance therapies were excluded. RESULTS: Thirty-three trials were included. For women with high-risk profiles that would contraindicate upfront cytoreductive surgery, neoadjuvant chemotherapy can be an option. In the post-surgical adjuvant setting, the three-weekly regimen consisting of paclitaxel and carboplatin remains the standard of care. Docetaxel may be offered to those who are unable to tolerate paclitaxel. Intraperitoneal cisplatin and paclitaxel increased OS for stage III optimally debulked women (GOG 172). The intraperitoneal regimens in GOG 252 offered no survival benefit and some harms in terms of toxicity and quality of life. CONCLUSIONS: There is no evidence to support adding a third agent to the standard carboplatin and paclitaxel. Results of the iPocc study will clarify the role of intraperitoneal chemotherapy.

Stp1 Loss of Function Promotes ß-Lactam Resistance in Staphylococcus aureus That Is Independent of Classical Genes.

ß-Lactam resistance in Staphylococcus aureus limits treatment options. Stp1 and Stk1, a serine-threonine phosphatase and kinase, respectively, mediate serine-threonine kinase (STK) signaling. Loss-of-function point mutations in stp1 were detected among laboratory-passaged ß-lactam-resistant S. aureus strains lacking mecA and blaZ, the major determinants of ß-lactam resistance in the bacteria. Loss of Stp1 function facilitates ß-lactam resistance of the bacteria.

Intracellular cholesterol biosynthesis in enchondroma and chondrosarcoma.

Enchondroma and chondrosarcoma are the most common benign and malignant cartilaginous neoplasms. Mutations in isocitrate dehydrogenase 1 and 2 (IDH1/2) are present in the majority of these tumors. We performed RNA-seq analysis on chondrocytes from Col2a1Cre;Idh1LSL/+ animals and found that genes implied in cholesterol synthesis pathway were significantly upregulated in the mutant chondrocytes. We examined the phenotypic effect of inhibiting intracellular cholesterol biosynthesis on enchondroma formation by conditionally deleting SCAP (sterol regulatory element-binding protein cleavage-activating protein), a protein activating intracellular cholesterol synthesis, in IDH1 mutant mice. We found fewer enchondromas in animals lacking SCAP. Furthermore, in chondrosarcomas, pharmacological inhibition of intracellular cholesterol synthesis significantly reduced chondrosarcoma cell viability in vitro and suppressed tumor growth in vivo. Taken together, these data suggest that intracellular cholesterol synthesis is a potential therapeutic target for enchondromas and chondrosarcomas.

Macrophage cells secrete factors including LRP1 that orchestrate the rejuvenation of bone repair in mice.

The pace of repair declines with age and, while exposure to a young circulation can rejuvenate fracture repair, the cell types and factors responsible for rejuvenation are unknown. Here we report that young macrophage cells produce factors that promote osteoblast differentiation of old bone marrow stromal cells. Heterochronic parabiosis exploiting young mice in which macrophages can be depleted and fractionated bone marrow transplantation experiments show that young macrophages rejuvenate fracture repair, and old macrophage cells slow healing in young mice. Proteomic analysis of the secretomes identify differential proteins secreted between old and young macrophages, such as low-density lipoprotein receptor-related protein 1 (Lrp1). Lrp1 is produced by young cells, and depleting Lrp1 abrogates the ability to rejuvenate fracture repair, while treating old mice with recombinant Lrp1 improves fracture healing. Macrophages and proteins they secrete orchestrate the fracture repair process, and young cells produce proteins that rejuvenate fracture repair in mice.

68Ga PET Imaging in Patients With Neuroendocrine Tumors: A Systematic Review and Meta-analysis.

PURPOSE: The aim of this study was to systematically review the literature to assess the role of Ga PET imaging in neuroendocrine tumors (NETs). MATERIALS AND METHODS: The literature was searched using MEDLINE, EMBASE, and Cochrane Database of Systematic Reviews databases through OVID. Studies comparing PET or PET/CT with conventional imaging in the initial diagnosis, staging and restaging, assessment of treatment response, and routine surveillance of NETs were deemed eligible for inclusion. Risk of bias and applicability concerns were assessed using the Quality Assessment of Diagnostic Accuracy Studies tool. RESULTS: Twenty-two studies met the inclusion criteria. For the initial diagnosis of NETs, PET or PET/CT had a pooled sensitivity of 91% (95% confidence interval [CI], 85%-94%) and a pooled specificity of 94% (95% CI, 86%-98%). In the setting of staging and restaging, the sensitivity of PET or PET/CT for detecting primary and/or metastatic lesions ranged from 78.3% to 100%, whereas specificity ranged from 83% to 100%. Change in management occurred in 45% (95% CI, 36%-55%) of the cases, with majority of the changes involving surgical planning and patient selection for peptide receptor radionuclide therapy. CONCLUSIONS: Ga PET or PET/CT is recommended for initial diagnosis where conventional testing remained equivocal, for staging of patients with localized primary and/or limited metastasis where definitive surgery is planned, to determine somatostatin receptor status and suitability for peptide receptor radionuclide therapy, and for staging of patients where detection of occult disease will alter treatment options and decision making.

A Metabolomics Pilot Study on Desmoid Tumors and Novel Drug Candidates.

Desmoid tumors (aggressive fibromatosis) are locally invasive soft tissue tumors that lack the ability to metastasize. There are no directed therapies or standard treatment plan, and chemotherapeutics, radiation, and surgery often have temporary effects. The majority of desmoid tumors are related to T41A and S45F mutations of the beta-catenin encoding gene (CTNNB1). Using broad spectrum metabolomics, differences were investigated between paired normal fibroblast and desmoid tumor cells from affected patients. There were differences identified, also, in the metabolomics profiles associated with the two beta-catenin mutations, T41A and S45F. Ongoing drug screening has identified currently available compounds which inhibited desmoid tumor cellular growth by more than 50% but did not affect normal fibroblast proliferation. Two drugs were investigated in this study, and Dasatinib and FAK Inhibitor 14 treatments resulted in unique metabolomics profiles for the normal fibroblast and desmoid tumor cells, in addition to the T41A and S45F. The biochemical pathways that differentiated the cell lines were aminoacyl-tRNA biosynthesis in mitochondria and cytoplasm and signal transduction amino acid-dependent mTORC1 activation. This study provides preliminary understanding of the metabolic differences of paired normal and desmoid tumors cells, their response to desmoid tumor therapeutics, and new pathways to target for therapy.

PET imaging in anal canal cancer: a systematic review and meta-analysis.

OBJECTIVE: The aim of this study was to systematically review the literature to synthesize and summarize the evidence surrounding the clinical utility of positron emission tomography (PET) imaging in patients with anal canal cancer. METHODS: The literature was searched using MEDLINE, EMBASE and Cochrane Database of Systematic Reviews databases. Studies comparing PET or PET/CT with conventional imaging in the staging, response evaluation and follow-up of anal canal cancer were deemed eligible for inclusion. RESULTS: 17 studies met the inclusion criteria. For the detection of primary tumour in situ, the pooled sensitivity was 99% for PET or PET/CT and 67% for CT. For the detection of inguinal lymph nodes, PET/CT had an overall sensitivity of 93% and specificity of 76%. PET or PET/CT upstaged 5.1 to 37.5% of patients and downstaged 8.2 to 26.7% of patients. Treatment plans were modified in 12.5 to 59.3% of patients, which consisted mainly of radiotherapy dose or field changes. Complete response on PET or PET/CT is a good prognostic factor for overall and progression-free survival. CONCLUSIONS: PET/CT seems to add value to conventional imaging in the initial staging of patients with T2-4 disease but further high-quality research is required to validate this. There is insufficient evidence at this time to recommend a routine use of PET/CT in the assessment of treatment response or follow-up. Advances in knowledge: PET/CT appears to alter the disease stage and management in a meaningful number of patients to justify its use as part of staging investigations in locally advanced cases.

Comparison of 18F-FDG-PET/CT and 18F-FDG-PET/MR imaging in oncology: a systematic review.

OBJECTIVE: The aim of this study was to systematically review the literature to evaluate the clinical performance of integrated 18F-FDG PET/MR as compared with 18F-FDG PET/CT in oncologic imaging. METHODS: The literature was searched using MEDLINE and EMBASE via OVID. Studies comparing the diagnostic accuracy of integrated 18F-FDG PET/MR and 18F-FDG PET/CT in the diagnosis, staging/restaging, assessment of treatment response, or evaluation of metastasis in patients with suspected or diagnosed cancers were deemed eligible for inclusion. Risk of bias and applicability concerns were assessed using the QUADAS-2 tool. RESULTS: Twenty studies met the inclusion criteria. The overall quality of the studies was rated favorably with bias or applicability concerns in a few studies. Our review suggests that 18F-FDG PET/MR performs comparably to 18F-FDG PET/CT in the detection of local lymph node and distant metastases and superiorly in determining the local extent of tumor. SUV obtained from 18F-FDG PET/MR correlated highly with those obtained from 18F-FDG PET/CT. CONCLUSIONS: Based on early evidence, 18F-FDG PET/MR is comparable to 18F-FDG PET/CT in the clinical scenarios examined in this review. The potential for interchangeability of 18F-FDG PET/MR with 18F-FDG PET/CT will vary by indication and the body site that is being imaged, with PET scanners integrated with MRI predicted to provide greater detail in the evaluation of local tumor extent, where 18F-FDG PET/CT can be limited.

Regulation of Cholesterol Homeostasis by Hedgehog Signaling in Osteoarthritic Cartilage.

OBJECTIVE: With no effective therapies to attenuate cartilage degeneration in osteoarthritis (OA), the result is pain and disability. Activation of hedgehog (HH) signaling causes changes related to the progression of OA, with higher levels of Gli-mediated transcriptional activation associated with increased disease severity. To elucidate the mechanism through which this occurs, this study sought to identify genes regulated by HH signaling in human OA chondrocytes. METHODS: Using human OA cartilage samples, microarray analyses were performed to detect changes in gene expression when the HH pathway was modulated. Results were analyzed for differentially expressed genes, grouped into functional networks, and validated in independent samples. To investigate the effects of chondrocyte-specific sterol accumulation, we generated mice lacking Insig1 and Insig2, which are major negative regulators of cholesterol homeostasis, under Col2a1 regulatory elements. RESULTS: HH signaling was found to regulate genes that govern cholesterol homeostasis, and this led to alterations in cholesterol accumulation in chondrocytes. A higher level of Gli-mediated transcription resulted in accumulation of intracellular cholesterol. In genetically modified mice, chondrocyte-specific cholesterol accumulation was associated with an OA phenotype. Reducing cholesterol accumulation attenuated the severity of OA in mice in vivo and decreased the expression of proteases in human OA cartilage in vitro. CONCLUSION: HH signaling regulates cholesterol homeostasis in chondrocytes, and intracellular cholesterol accumulation contributes to the severity of OA. Our findings have therapeutic implications, since reduction of HH signaling reversed cholesterol accumulation and statin treatment attenuated cartilage degeneration.

The Utility of Positron Emission Tomography in Epilepsy.

The role of fludeoxyglucose F 18 positron emission tomography (PET) in the presurgical evaluation of patients with medically intractable epilepsy continues to be refined. The purpose of this study was to systematically review the literature to assess the diagnostic accuracy and utility of PET in this setting. Thirty-nine studies were identified through MEDLINE and EMBASE databases that met the inclusion criteria. In adult patients, PET hypometabolism showed a 56 to 90% agreement with seizure onset localized by intracranial electroencephalogram (pediatric: 21 to 86%). In temporal lobe epilepsy patients with good surgical outcome, PET displayed moderate to high sensitivity in localizing the seizure focus (range: 71 to 89%). The sensitivity increased by 8 to 23% when PET results were combined with magnetic resonance imaging or electroencephalogram. PET has been shown to affect patient management by improving the guidance of intracranial electrodes placement, altering the decision to perform surgery, or excluding patients from further evaluation.

Mutant IDH is sufficient to initiate enchondromatosis in mice.

Enchondromas are benign cartilage tumors and precursors to malignant chondrosarcomas. Somatic mutations in the isocitrate dehydrogenase genes (IDH1 and IDH2) are present in the majority of these tumor types. How these mutations cause enchondromas is unclear. Here, we identified the spectrum of IDH mutations in human enchondromas and chondrosarcomas and studied their effects in mice. A broad range of mutations was identified, including the previously unreported IDH1-R132Q mutation. These mutations harbored enzymatic activity to catalyze α-ketoglutarate to d-2-hydroxyglutarate (d-2HG). Mice expressing Idh1-R132Q in one allele in cells expressing type 2 collagen showed a disordered growth plate, with persistence of type X-expressing chondrocytes. Chondrocyte cell cultures from these animals or controls showed that there was an increase in proliferation and expression of genes characteristic of hypertrophic chondrocytes with expression of Idh1-R132Q or 2HG treatment. Col2a1-Cre;Idh1-R132Q mutant knock-in mice (mutant allele expressed in chondrocytes) did not survive after the neonatal stage. Col2a1-Cre/ERT2;Idh1-R132 mutant conditional knock-in mice, in which Cre was induced by tamoxifen after weaning, developed multiple enchondroma-like lesions. Taken together, these data show that mutant IDH or d-2HG causes persistence of chondrocytes, giving rise to rests of growth-plate cells that persist in the bone as enchondromas.

Development of a sensitive in vitro assay to quantify the biological activity of pro-inflammatory phorbol esters in Jatropha oil.

New health safety concerns may arise from the increasing production and use of Jatropha oil, a biodiesel feedstock that also contains toxic, pro-inflammatory, and co-carcinogenic phorbol esters. Based on the exceptional sensitivity of Madin-Darby canine kidney (MDCK) cells to the model phorbol ester 12-O-tetradecanoylphorbol-13-acetate (TPA), a robust bioassay was developed to quantify the biological activity of Jatropha phorbol esters directly in oil, without sample extraction. We first verified that the characteristic response of MDCK cells to TPA was also observed following direct exposure to phorbol esters in Jatropha oil. We further confirmed that similarly to TPA, Jatropha oil's phorbol esters can activate protein kinase C (PKC). We then assessed the transcriptional response of MDCK cells to Jatropha oil exposure by measuring the expression of cyclooxygenase-2 (COX-2), a gene involved in inflammatory processes which is strongly upregulated following PKC activation. Based on the parameterization of a TPA dose-response curve, the transcriptional response of MDCK cells to Jatropha oil exposure was expressed in term of TPA toxic equivalent (TEQ), a convenient metric to report the inflammatory potential of complex mixtures. The sensitive bioassay described in this manuscript may prove useful for risk assessment, as it provides a quantitative method and a convenient metric to report the inflammatory potential of phorbol esters in Jatropha oil. This bioassay may also be adapted for the detection of bioactive phorbol esters in other matrices.