Organoid models of breathing disorders reveal patterning defect of hindbrain neurons caused by PHOX2B-PARMs.

Retrotrapezoid nucleus (RTN) neurons in the brainstem regulate the ventilatory response to hypercarbia. It is unclear how PHOX2B-polyalanine repeat mutations (PHOX2B-PARMs) alter the function of PHOX2B and perturb the formation of RTN neurons. Here, we generated human brainstem organoids (HBSOs) with RTN-like neurons from human pluripotent stem cells. Single-cell transcriptomics revealed that expression of PHOX2B+7Ala PARM alters the differentiation trajectories of the hindbrain neurons and hampers the formation of the RTN-like neurons in HBSOs. With the unguided cerebral organoids (HCOs), PHOX2B+7Ala PARM interrupted the patterning of PHOX2B+ neurons with dysregulation of Hedgehog pathway and HOX genes. With complementary use of HBSOs and HCOs with a patient and two mutant induced pluripotent stem cell lines carrying different polyalanine repetition in PHOX2B, we further defined the association between the length of polyalanine repetition and malformation of RTN-respiratory center and demonstrated the potential toxic gain of function of PHOX2B-PARMs, highlighting the uniqueness of these organoid models for disease modeling.

Transcriptomics of Hirschsprung disease patient-derived enteric neural crest cells reveals a role for oxidative phosphorylation.

Hirschsprung disease is characterized by the absence of enteric neurons caused by the defects of enteric neural crest cells, leading to intestinal obstruction. Here, using induced pluripotent stem cell-based models of Hirschsprung and single-cell transcriptomic analysis, we identify a gene set of 118 genes commonly dysregulated in all patient enteric neural crest cells, and suggest HDAC1 may be a key regulator of these genes. Furthermore, upregulation of RNA splicing mediators and enhanced alternative splicing events are associated with severe form of Hirschsprung. In particular, the higher inclusion rate of exon 9 in PTBP1 and the perturbed expression of a PTBP1-target, PKM, are significantly enriched in these patient cells, and associated with the defective oxidative phosphorylation and impaired neurogenesis. Hedgehog-induced oxidative phosphorylation significantly enhances the survival and differentiation capacity of patient cells. In sum, we define various factors associated with Hirschsprung pathogenesis and demonstrate the implications of oxidative phosphorylation in enteric neural crest development and HSCR pathogenesis.

Longitudinal comparison of direct medical cost, radiological and health-related quality of life treatment outcomes between traditional growing rods and magnetically controlled growing rods from preoperative to maturity.

BACKGROUND: Magnetically controlled growing rods (MCGR) have replaced traditional growing rods (TGR) in the past decade, however, a comparison of their direct costs and treatment outcomes based on real longitudinal data is lacking. This study aims to compare the direct cost and treatment outcomes between TGR and MCGR, whilst incorporating complications, reoperations and changes in health-related quality of life (HRQoL) throughout the entire treatment course. METHODS: Patients with early onset scoliosis (EOS) who underwent initial growing rod surgery between 2003 and 2016 at a tertiary scoliosis clinic were studied with longitudinal data. Accumulated direct medical costs were calculated based on the unit cost of surgeries of each TGR and MCGR, costs incurred for any rod exchange or remedial surgery for post-operative complication. Treatment outcomes were evaluated via: Patient's HRQoL using SRS-22r questionnaire, and radiological parameters (including major curve correction, spine length gains, spinal balance) throughout the treatment until maturity. RESULTS: A total of 27 EOS patients (16 MCGR, 11 TGR) were studied. Total direct cost of index surgery for MCGR was HKD$223,108 versus lower cost of HKD$135,184 for TGR (p < 0.001). At 2-3 years post-index surgery, accumulative total direct medical cost of MCGR and TGR became most comparable (TGR:MCGR ratio = 1.010) and had reached neutrality between the two groups since. Radiological parameters had no intergroup differences at maturity. For HRQoL, TGR group had shown the trend of less pain (domain score mean difference: 0.53, p = 0.024) post-index surgery and better self-appearance (domain score mean difference: 1.08, p = 0.017) before fusion. Higher satisfaction with treatment (domain score mean difference: 0.76, p = 0.029) was demonstrated by TGR patients at fusion/maturity. MCGR had negative (rs = -0.693) versus TGR's positive (rs = 0.989) correlations (p < 0.05) of cost and SRS-22r total scores at 2-3 years post-index surgery. CONCLUSIONS: From index surgery to maturity, TGR demonstrated better satisfaction with treatment by patients and comparable overall HRQoL with MCGR during the treatment course, as MCGR did not show apparent benefit despite less surgeries and cost neutrality between the two groups at 2-3 years post-index surgery.

Ciliary protein Kif7 regulates Gli and Ezh2 for initiating the neuronal differentiation of enteric neural crest cells during development.

Gastrointestinal motility disorders occur frequently in patients with ciliopathy, but the underlying genetic link is unclear. The ciliary protein Kif7 can positively or negatively regulate Hedgehog signaling in different cellular contexts. Mice with neural crest cell (NCC)­specific Kif7 deficiency show a marked reduction of enteric NOS+ inhibitory neurons. Malformation of enteric nervous system (ENS) causes growth retardation and gut motility defect in mice. Mechanistically, Kif7 inhibits Gli2 in enteric NCCs (ENCCs), where Gli2 positively regulates the expression of Ezh2 by inhibiting the miR124-mediated suppression. In developing ENCCs, Ezh2 is a master regulator of 102 core genes underlying ENCC differentiation. Deletion of Gli2 or inhibition of Ezh2 favors the neurogenic lineage differentiation of mouse and human ENCCs and rescues the ENS defects of Kif7 mutants. In summary, Hedgehog signal, via Kif7-Gli-Ezh2, controls the timely expressions of the core genes to mediate the differentiation of ENCCs.

Multidisciplinary programme for rehabilitation of chronic low back pain - factors predicting successful return to work.

BACKGROUND: There are no clear indicators for predicting return to work for patients with chronic low back pain (LBP). We aim to report the outcomes of a 14-week multidisciplinary programme targeting patients with chronic LBP who failed conventional physiotherapy to provide functional rehabilitation. Also, this study will identify factors predicting successful return to work (RTW). METHODS: A collected cohort of patients with chronic LBP was consecutively enrolled into the programme from 1996 to 2014. All recruited patients failed to RTW despite at least 3 months of conservative treatment. Patient underwent weekly multidisciplinary sessions with physiotherapists, occupational therapists and clinical psychologists. Patient perceived function was considered the primary outcome of the programme. Patients were assessed for their sitting, standing and walking tolerance. Oswestry Disability Index (ODI) and Spinal Function Sort Score (SFSS) were used to assess patient perceived disability. RESULTS: One hundred and fifty-eight patients were recruited. After the programme, statistically significant improvement was found in ODI (47.5 to 45.0, p = 0.01) and SFSS (98.0 to 109.5, p <  0.001). There was statistically significant improvement (p <  0.01) in sitting, standing, walking tolerance and straight leg raise tests. 47.4% of the patients were able to meet their work demand. Multivariate logistic regression model (R2 = 59.5%, χ2 (9) = 85.640, p <  0.001) demonstrated that lower initial job demand level and higher patient-perceived back function correlated with greater likelihood of returning to work. CONCLUSION: The results of this study may support the use of this multidisciplinary programme to improve patient function and return to work.

Whole-genome analysis of noncoding genetic variations identifies multiscale regulatory element perturbations associated with Hirschsprung disease.

It is widely recognized that noncoding genetic variants play important roles in many human diseases, but there are multiple challenges that hinder the identification of functional disease-associated noncoding variants. The number of noncoding variants can be many times that of coding variants; many of them are not functional but in linkage disequilibrium with the functional ones; different variants can have epistatic effects; different variants can affect the same genes or pathways in different individuals; and some variants are related to each other not by affecting the same gene but by affecting the binding of the same upstream regulator. To overcome these difficulties, we propose a novel analysis framework that considers convergent impacts of different genetic variants on protein binding, which provides multiscale information about disease-associated perturbations of regulatory elements, genes, and pathways. Applying it to our whole-genome sequencing data of 918 short-segment Hirschsprung disease patients and matched controls, we identify various novel genes not detected by standard single-variant and region-based tests, functionally centering on neural crest migration and development. Our framework also identifies upstream regulators whose binding is influenced by the noncoding variants. Using human neural crest cells, we confirm cell stage-specific regulatory roles of three top novel regulatory elements on our list, respectively in the RET, RASGEF1A, and PIK3C2B loci. In the PIK3C2B regulatory element, we further show that a noncoding variant found only in the patients affects the binding of the gliogenesis regulator NFIA, with a corresponding up-regulation of multiple genes in the same topologically associating domain.

Cost analysis comparison between conventional microsurgical decompression and full-endoscopic interlaminar decompression for lumbar spinal stenosis surgery.

BACKGROUND: Two of the most commonly used techniques for treatment of lumbar spinal stenosis (LSS) include full-endoscopic interlaminar decompression (MIS) and conventional microsurgical decompression (CD). Although these procedures have proven efficacy for relief of stenotic symptoms, in this age of increased concerns for healthcare cost, weighing the respective accumulative costs is essential for deciding which approach to adopt. The aim of this study is to perform a cost analysis comparison between MIS and CD for LSS. METHODS: A decision analysis model comparing MIS and CD for patients with LSS over a 1-year time horizon was conducted. Relevant unit costs associated with each surgical procedure and each possible complication treatment were estimated. Regarding the respective complication rates for each procedure, data was retrieved from the literature. Reoperation was considered for epidural hematoma, inadequate decompression or iatrogenic instability requiring fusion. Nonoperative treatment for complications like infection was also considered. RESULTS: The average total costs for MIS and CD were found to be HKD$54,863 and HKD$52,748 respectively. Both procedures carried similar costs in terms of hospitalization, radiology and routine follow-up visits. A 3.9% (HKD$2,115) difference in total cost was largely due to the differences in cost of surgery and complications. MIS costs 5.7% more than CD for an operation but was 28.1% less costly than MIS for complications. CONCLUSIONS: Given the similar clinical effectiveness of either procedure and only a small difference in overall cost, our findings suggest that surgeons should perform the procedure that they are competent with which guarantees adequacy of decompression.

Activation of Hedgehog Signaling Promotes Development of Mouse and Human Enteric Neural Crest Cells, Based on Single-Cell Transcriptome Analyses.

BACKGROUND & AIMS: It has been a challenge to develop fully functioning cells from human pluripotent stem cells (hPSCs). We investigated how activation of hedgehog signaling regulates derivation of enteric neural crest (NC) cells from hPSCs. METHODS: We analyzed transcriptomes of mouse and hPSC-derived enteric NCs using single-cell RNA sequencing (scRNA-seq) to identify the changes in expression associated with lineage differentiation. Intestine tissues were collected from Tg(GBS-GFP), Sufuf/f; Wnt1-cre, Ptch1+/-, and Gli3Δ699/Δ699 mice and analyzed by flow cytometry and immunofluorescence for levels of messenger RNAs encoding factors in the hedgehog signaling pathway during differentiation of enteric NCs. Human NC cells (HNK-1+p75NTR+) were derived from IMR90 and UE02302 hPSC lines. hPSCs were incubated with a hedgehog agonist (smoothened agonist [SAG]) and antagonists (cyclopamine) and analyzed for differentiation. hPSC-based innervated colonic organoids were derived from these hPSC lines and analyzed by immunofluorescence and neuromuscular coupling assay for expression of neuronal subtype markers and assessment of the functional maturity of the hPSC-derived neurons, respectively. RESULTS: Single-cell RNA sequencing analysis showed that neural fate acquisition by human and mouse enteric NC cells requires reduced expression of NC- and cell cycle-specific genes and up-regulation of neuronal or glial lineage-specific genes. Activation of the hedgehog pathway was associated with progression of mouse enteric NCs to the more mature state along the neuronal and glial lineage differentiation trajectories. Activation of the hedgehog pathway promoted development of cultured hPSCs into NCs of greater neurogenic potential by activating expression of genes in the neurogenic lineage. The hedgehog agonist increased differentiation of hPSCs into cells of the neuronal lineage by up-regulating expression of GLI2 target genes, including INSM1, NHLH1, and various bHLH family members. The hedgehog agonist increased expression of late neuronal markers and neuronal activities in hPSC-derived neurons. CONCLUSIONS: In enteric NCs from humans and mice, activation of hedgehog signaling promotes differentiation into neurons by promoting cell-state transition, expression of genes in the neurogenic lineage, and functional maturity of enteric neurons.

Identification of Genes Associated With Hirschsprung Disease, Based on Whole-Genome Sequence Analysis, and Potential Effects on Enteric Nervous System Development.

BACKGROUND & AIMS: Hirschsprung disease, or congenital aganglionosis, is believed to be oligogenic-that is, caused by multiple genetic factors. We performed whole-genome sequence analyses of patients with Hirschsprung disease to identify genetic factors that contribute to disease development and analyzed the functional effects of these variants. METHODS: We performed whole-genome sequence analyses of 443 patients with short-segment disease, recruited from hospitals in China and Vietnam, and 493 ethnically matched individuals without Hirschsprung disease (controls). We performed genome-wide association analyses and gene-based rare-variant burden tests to identify rare and common disease-associated variants and study their interactions. We obtained induced pluripotent stem cell (iPSC) lines from 4 patients with Hirschsprung disease and 2 control individuals, and we used these to generate enteric neural crest cells for transcriptomic analyses. We assessed the neuronal lineage differentiation capability of iPSC-derived enteric neural crest cells using an in vitro differentiation assay. RESULTS: We identified 4 susceptibility loci, including 1 in the phospholipase D1 gene (PLD1) (P = 7.4 × 10-7). The patients had a significant excess of rare protein-altering variants in genes previously associated with Hirschsprung disease and in the ß-secretase 2 gene (BACE2) (P = 2.9 × 10-6). The epistatic effects of common and rare variants across these loci provided a sensitized background that increased risk for the disease. In studies of the iPSCs, we observed common and distinct pathways associated with variants in RET that affect risk. In functional assays, we found variants in BACE2 to protect enteric neurons from apoptosis. We propose that alterations in BACE1 signaling via amyloid ß precursor protein and BACE2 contribute to pathogenesis of Hirschsprung disease. CONCLUSIONS: In whole-genome sequence analyses of patients with Hirschsprung disease, we identified rare and common variants associated with disease risk. Using iPSC cells, we discovered some functional effects of these variants.

Responsiveness of the EuroQoL 5-dimension (EQ-5D) in adolescent idiopathic scoliosis.

PURPOSE: To test the responsiveness of the EuroQoL 5-dimension (EQ-5D) utility scores for adolescent idiopathic scoliosis (AIS). METHODS: A baseline sample of 227 AIS patients was recruited between August and October 2015, and was surveyed prospectively to 9-12 months follow-up. EQ-5D-5L utility scores were derived using a two-step approach: (1) cross-walking from five-level responses to three-level responses and (2) applying the EQ-5D-3L Chinese population value set. An anchor approach was adopted to assess the responsiveness of EQ-5D. Effect size statistics (standardized effect size and standardized response mean) and independent t test were used to assess the responsiveness, as well as to analyze the ability of measures to detect score changes with global health condition changes or discriminate between the worsened and unchanged/improved groups. RESULTS: Approximately two-thirds of follow-up patients (64.2%) reported no change in global health condition based on the self-reported health anchor, whilst 4.6 and 31.3% of patients rated worse and better in current health condition compared to baseline, respectively. In the subgroup where health worsened, EQ-5D utility scores were responsive to detect negative changes. EQ-5D utility scores had slight improvement in the group where health improved, despite a high mean score of 0.92 at baseline. Neither statistical significance nor moderate-large effect size was observed in mean changes among unchanged group. Responsiveness property of the EQ-5D utility score was generally satisfactory with respect to each health condition group. CONCLUSIONS: EQ-5D is found to be able to capture positive changes, and responsive in detecting important clinical changes in the improved group of this AIS population.

Psychometric Validation of the Adapted Traditional Chinese (Hong Kong) Version of the Japanese Orthopaedic Association Cervical Myelopathy Evaluation Questionnaire (JOACMEQ).

STUDY DESIGN: A prospective questionnaire translation and validation. OBJECTIVE: The aim of this study was to translate and cross-culturally adapt the Japanese Orthopaedic Association Cervical Myelopathy Evaluation Questionnaire (JOACMEQ) into Traditional Chinese (Hong Kong) and to assess its validity, reliability, and sensitivity for differentiating cervical myelopathy (CM) and presence of acute neck/shoulder pain. SUMMARY OF BACKGROUND DATA: CM frequently presents with various symptoms affecting patients' quality of life. Hence, a patient-oriented instrument such as JOACMEQ is necessary to assess patient-perceived outcomes of CM treatment. METHODS: The English version of JOACMEQ was translated and adapted using double forward and single backward translations. The translated JOACMEQ was administered to patients with suspected CM, followed by the Traditional Chinese (Hong Kong) version of the Neck Disability Index (NDI), EuroQol five-dimension five-level (EQ-5D-5L), and Short Form-12 version 2 (SF-12v2) questionnaires. Construct validity of the domains was assessed using Spearman correlation test against domains with similar constructs. Internal consistency was assessed by Cronbach's alpha. Sensitivity of the adapted JOACMEQ was determined by known group comparisons. RESULTS: A total of 100 patients were recruited. Psychometric testing of the translated JOACMEQ demonstrated an excellent overall internal consistency with Cronbach's α > 0.9, and good internal consistency of Lower Extremity Function (0.823) and Quality of Life (0.875) domains. Score of all domains of the translated JOACMEQ had significant correlations (P < 0.01-0.05) with nearly all domains of SF-12v2 and with both NDI and EQ-5D-5L scores. JOACMEQ was sensitive in detecting differences (P < 0.001) between subjects who had CM and those without, and also between those patients with/without CM experiencing current neck/shoulder pain. CONCLUSION: Our translated JOACMEQ has satisfactory psychometric properties, including adequate clinical and construct validity, and internal consistency in patients with suspected/diagnosed CM and can differentiate between those with/without pain. It is demonstrated as a sensitive outcome measure for CM and neck/shoulder pain. LEVEL OF EVIDENCE: 2.

Correction of Hirschsprung-Associated Mutations in Human Induced Pluripotent Stem Cells Via Clustered Regularly Interspaced Short Palindromic Repeats/Cas9, Restores Neural Crest Cell Function.

BACKGROUND & AIMS: Hirschsprung disease is caused by failure of enteric neural crest cells (ENCCs) to fully colonize the bowel, leading to bowel obstruction and megacolon. Heterozygous mutations in the coding region of the RET gene cause a severe form of Hirschsprung disease (total colonic aganglionosis). However, 80% of HSCR patients have short-segment Hirschsprung disease (S-HSCR), which has not been associated with genetic factors. We sought to identify mutations associated with S-HSCR, and used the clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 gene editing system to determine how mutations affect ENCC function. METHODS: We created induced pluripotent stem cell (iPSC) lines from 1 patient with total colonic aganglionosis (with the G731del mutation in RET) and from 2 patients with S-HSCR (without a RET mutation), as well as RET+/- and RET-/- iPSCs. IMR90-iPSC cells were used as the control cell line. Migration and differentiation capacities of iPSC-derived ENCCs were analyzed in differentiation and migration assays. We searched for mutation(s) associated with S-HSCR by combining genetic and transcriptome data from patient blood- and iPSC-derived ENCCs, respectively. Mutations in the iPSCs were corrected using the CRISPR/Cas9 system. RESULTS: ENCCs derived from all iPSC lines, but not control iPSCs, had defects in migration and neuronal lineage differentiation. RET mutations were associated with differentiation and migration defects of ENCCs in vitro. Genetic and transcriptome analyses associated a mutation in the vinculin gene (VCL M209L) with S-HSCR. CRISPR/Cas9 correction of the RET G731del and VCL M209L mutations in iPSCs restored the differentiation and migration capacities of ENCCs. CONCLUSIONS: We identified mutations in VCL associated with S-HSCR. Correction of this mutation in iPSC using CRISPR/Cas9 editing, as well as the RET G731del mutation that causes Hirschsprung disease with total colonic aganglionosis, restored ENCC function. Our study demonstrates how human iPSCs can be used to identify disease-associated mutations and determine how they affect cell functions and contribute to pathogenesis.

Dysregulation of clathrin promotes thyroid cell growth and contributes to multinodular goiter pathogenesis.

A germline mutation (A339V) in thyroid transcription factor-1 (TITF1/NKX2.1) was shown to be associated with multinodular goiter (MNG) and papillary thyroid carcinoma (PTC) pathogenesis. The overexpression of A339V TTF1 significantly promoted hormone-independent growth of the normal thyroid cells, representing a cause of MNG and/or PTC. Nevertheless, the underlying mechanism still remains unclear. In this study, we used liquid chromatography (LC)-tandem mass spectrometry (MS/MS)-based shotgun proteomics comparing the global protein expression profiles of normal thyroid cells (PCCL3) that overexpressed the wild-type or A339V TTF1 to identify key proteins implicated in this process. Proteomic pathway analysis revealed that the aberrant activation of epidermal growth factor (EGF) signaling is significantly associated with the overexpression of A339V TTF1 in PCCL3, and clathrin heavy chain (Chc) is the most significantly up-regulated protein of the pathway. Intriguingly, dysregulated Chc expression facilitated a nuclear accumulation of pStat3, leading to an enhanced cell proliferation of the A339V clones. Down-regulation and abrogation of Chc-mediated cellular trafficking, respectively, by knocking-down Chc and ectopic expression of a dominant-negative (DN) form of Chc could significantly reduce the nuclear pStat3 and rescue the aberrant cell proliferation of the A339V clones. Subsequent expression analysis further revealed that CHC and pSTAT3 are co-overexpressed in 66.7% (10/15) MNG. Taken together, our results suggest that the A339V TTF1 mutant protein up-regulates the cellular expression of Chc, resulting in a constitutive activation of Stat3 pathway, and prompting the aberrant growth of thyroid cells. This extensive growth signal may promote the development of MNG.

Hedgehog/Notch-induced premature gliogenesis represents a new disease mechanism for Hirschsprung disease in mice and humans.

Hirschsprung (HSCR) disease is a complex genetic disorder attributed to a failure of the enteric neural crest cells (ENCCs) to form ganglia in the hindgut. Hedgehog and Notch are implicated in mediating proliferation and differentiation of ENCCs. Nevertheless, how these signaling molecules may interact to mediate gut colonization by ENCCs and contribute to a primary etiology for HSCR are not known. Here, we report our pathway-based epistasis analysis of data generated by a genome-wide association study on HSCR disease, which indicates that specific genotype constellations of Patched (PTCH1) (which encodes a receptor for Hedgehog) and delta-like 3 (DLL3) (which encodes a receptor for Notch) SNPs confer higher risk to HSCR. Importantly, deletion of Ptch1 in mouse ENCCs induced robust Dll1 expression and activation of the Notch pathway, leading to premature gliogenesis and reduction of ENCC progenitors in mutant bowels. Dll1 integrated Hedgehog and Notch pathways to coordinate neuronal and glial cell differentiation during enteric nervous system development. In addition, Hedgehog-mediated gliogenesis was found to be highly conserved, such that Hedgehog was consistently able to promote gliogenesis of human neural crest-related precursors. Collectively, we defined PTCH1 and DLL3 as HSCR susceptibility genes and suggest that Hedgehog/Notch-induced premature gliogenesis may represent a new disease mechanism for HSCR.

Role of the RAS in pancreatic cancer.

Angiotensin II (Ang II), a main effector peptide of the renin-angiotensin system (RAS), mediates a hormonal action in the maintenance of blood pressure and electrolyte levels, and thus fluid homeostasis. Ang II also mediates paracrine, autocrine and/or intracrine actions in the control of various specific functions of diverse tissue organs. In the pancreas, Ang II exerts a growth promoting, angiogenic influence via the mediation of angiotensin II type 1 receptor (AT1R). Recent studies have implicated inappropriate activation of the local RAS in pancreatic cancer, including upregulation of AT1R and the angiotensin-converting enzyme (ACE), which consequently enhance Ang II-induced tumour activity. In addition to acting in a classical antihypertensive capacity, RAS blockers (AT1R blockers or ACE inhibitors) may yield protective effects against pancreatic cancer, a highly aggressive malignancy that is intrinsically resistant to radiotherapy and chemotherapy. Substantial experimental data from studies using cell and animal models of pancreatic cancer support the notion that RAS regulates tumour growth, angiogenesis, and metastasis; and a convergence of such findings suggests that pharmacological RAS blockade could have therapeutic potential in the management of pancreatic cancer. This review critically appraises the current research progress on the role of RAS in pancreatic cancer, and discusses the potential for developing drugs that target RAS for treatment of pancreatic cancer.

Bruceine D induces apoptosis in pancreatic adenocarcinoma cell line PANC-1 through the activation of p38-mitogen activated protein kinase.

Pancreatic cancer is a malignant disease with extremely high mortality. Our previous work found that Brucea javanica fruit possesses potent anti-pancreatic cancer activity. In the present study, brucein D (BD), a quassinoid found abundantly in B. javanica fruit, was evaluated for its anti-proliferative and apoptogenic actions. BD inhibited the growth of three pancreatic cancer cell lines, i.e., PANC-1, SW1990 and CAPAN-1, but exerted only modest cytotoxicity on non-tumorigenic Hs68 cells. Hoechst 33342 staining and Cell Death Detection ELISA(PLUS) assay revealed that BD-induced DNA fragmentation in PANC-1 cells. Moreover, subG1 phase was observed in the BD-treated cells. Western blot experiments indicated that BD exposure augmented caspase 3, 8, 9 and bak protein levels, while attenuating the expression of bcl-2. Furthermore, BD treatment promoted phosphorylation of p38-MAPK. The selective p38-MAPK inhibitor SB203580 effectively mitigated the BD-induced apoptosis in PANC-1 cells, suggesting that p38-MAPK signaling pathway was involved in the BD-induced apoptosis in pancreatic cancer cells. Taken together, our results provide experimental evidence to support the traditional use of B. javanica fruit in cancer treatment, and render BD a promising chemical candidate for further development into anti-pancreatic cancer agent.

Brucea javanica fruit induces cytotoxicity and apoptosis in pancreatic adenocarcinoma cell lines.

Brucea javanica fruit is thought to have anticancer properties in Chinese medicine and its extract has been shown to possess antiproliferative and pro-apoptotic activities on human carcinoma cells. In the present study we demonstrated for the first time that Fructus Bruceae extract exhibited cytotoxic effects on the three pancreatic adenocarcinoma cell lines, PANC-1, SW1990 and CAPAN-1; the effects were comparable to those exhibited by camptothecin in our culture system. In addition, Fructus Bruceae extract induced fragmentation of genomic DNA, as evidenced by Hoechst staining and the cell death detection ELISA(PLUS) assay. Western blot analysis further showed down-regulation of pro-caspase 3 protein expression, indicating that the observed cytotoxic effects of the extract were associated with induction of apoptosis. These findings are not only significant in the development of traditional Chinese medicine as an alternative treatment for pancreatic cancer, but also in the elucidation of the potential mechanism(s) of Fructus Bruceae extract in cancer therapy.