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1.  Hypoxia-inducible miR-182 enhances HIF1α signaling via targeting PHD2 and FIH1 in prostate cancer 
Scientific Reports  2015;5:12495.
Activation of hypoxia-inducible factor 1α (HIF1α) controls the transcription of genes governing angiogenesis under hypoxic condition during tumorigenesis. Here we show that hypoxia-responsive miR-182 is regulated by HIF1α at transcriptional level. Prolyl hydroxylase domain enzymes (PHD) and factor inhibiting HIF-1 (FIH1), negative regulators of HIF1 signaling, are direct targets of miR-182. Overexpression of miR-182 in prostate cancer cells led to a reduction of PHD2 and FIH1 expression and an increase in HIF1α level either under normoxic or hypoxic condition. Consistently, inhibition of miR-182 could increase PHD2 and FIH1 levels, thereby reducing the hypoxia-induced HIF1α expression. Matrigel plug assay showed that angiogenesis was increased by miR-182 overexpression, and vice versa. miR-182 overexpression in PC-3 prostate cancer xenografts decreased PHD2 and FIH1 expression, elevated HIF1α protein levels, and increased tumor size. Lastly, we revealed that the levels of both miR-182 and HIF1α were elevated, while the expression PHD2 and FIH1 was downregulated in a mouse model of prostate cancer. Together, our results suggest that the interplay between miR-182 and HIF1α could result in a sustained activation of HIF1α pathway, which might facilitate tumor cell adaption to hypoxic stress during prostate tumor progression.
doi:10.1038/srep12495
PMCID: PMC4513346  PMID: 26205124
2.  TGFβ1 secreted by cancer-associated fibroblasts induces epithelial-mesenchymal transition of bladder cancer cells through lncRNA-ZEB2NAT 
Scientific Reports  2015;5:11924.
Urinary bladder cancer (UBC) patients at muscle invasive stage have poor clinical outcome, due to high propensity for metastasis. Cancer-associated fibroblasts (CAFs), one of the principal constituents of the tumor stroma, play an important role in tumor development. However, it is unclear whether CAFs from UBC induce cell invasion and which signaling pathway is involved. Herein, we found that conditional medium from UBC CAFs (CAF-CM) enhanced the invasion of UBC cells. CAF-CM induced the epithelial-mesenchymal transition (EMT) by regulating expression levels of EMT-associated markers in UBC cells. Higher concentration of TGFβ1 in CAF-CM, comparing with the CM from adjacent normal fibroblast, led to phosphorylation of Smad2 in UBC cells. Additionally, inhibition of TGFβ1 signaling decreased the EMT-associated gene expression, and cancer cell invasion. Interestingly, a long non-coding RNA, ZEB2NAT, was demonstrated to be essential for this TGFβ1-dependent process. ZEB2NAT depletion reversed CAF-CM-induced EMT and invasion of cancer cells, as well as reduced the ZEB2 protein level. Consistently, TGFβ1 mRNA expression is positively correlated with ZEB2NAT transcript and ZEB2 protein levels in human bladder cancer specimens. Our data revealed a novel mechanism that CAFs induces EMT and invasion of human UBC cells through the TGFβ1-ZEB2NAT-ZEB2 axis.
doi:10.1038/srep11924
PMCID: PMC4495469  PMID: 26152796
3.  Human polymorphonuclear neutrophils specifically recognize and kill cancerous cells 
Oncoimmunology  2014;3(7):e950163.
Polymorphonuclear neutrophils (PMNs), the main effectors of the innate immune system, have rarely been considered as an anticancer therapeutic tool. However, recent investigations using animal models and preliminary clinical studies have highlighted the potential antitumor efficacy of PMNs. In the current study, we find that PMNs from some healthy donors naturally have potent cancer-killing activity against 4 different human cancer cell lines. The killing activity appears to be cancer cell-specific since PMNs did not kill primary normal epithelial cells or an immortalized breast epithelial cell line. Transfecting the immortalized mammary cells with plasmids expressing activated forms of the rat sarcoma viral oncogene homolog (Ras) and teratocarcinoma oncogene 21 (TC21) oncogenes was sufficient to provoke aggressive attack by PMNs. However, transfection with activated Ras-related C3 botulinum toxin substrate (Rac1) was ineffective, suggesting specificity in PMN-targeting of neoplastic cells. Furthermore, PMNs from lung cancer patients were also found to exhibit relatively poor cancer-killing activity compared to the cytolytic activity of the average healthy donor. Taken together, our results suggest that PMN-based treatment regimens may represent a paradigm shift in cancer immunotherapy that may be easily introduced into the clinic to benefit a subset of patients with PMN-vulnerable tumors.
doi:10.4161/15384101.2014.950163
PMCID: PMC4292216  PMID: 25610737
cytotoxicity; neutrophils; oncogene; tumor cells; BEN, benign ethnic neutropenia; DBL, proto-oncogene DBL; DPI, diphenyleneiodonium; E:T, effector:target; G-CSF, granulocyte-colony stimulating factor; GM-CSF, granulocyte macrophage-colony stimulating factor; GVHD, graft-versus-host disease; H-Ras, Harvey rat sarcoma viral oncogene homolog; mAb, monoclonal antibody; MEK, mitogen-activated protein kinase kinase; mTOR, mammalian target of rapamycin; NADPH, nicotinamide adenine dinucleotide phosphate; NBT, nitroblue tetrazolium; NSCLC, non-small cell lung carcinoma; PI3 kinase, phosphoinositide 3-kinase; PMN, polymorphonuclear neutrophils; Rac1, Ras-related C3 botulinum toxin substrate 1; RhoA, Ras homolog family member A; ROS, reactive oxygen species; TC-21, teratocarcinoma oncogene TC21; TGFβ, transforming growth factor
4.  Similar Impacts of the Interaural Delay and Interaural Correlation on Binaural Gap Detection 
PLoS ONE  2015;10(6):e0126342.
The subjective representation of the sounds delivered to the two ears of a human listener is closely associated with the interaural delay and correlation of these two-ear sounds. When the two-ear sounds, e.g., arbitrary noises, arrive simultaneously, the single auditory image of the binaurally identical noises becomes increasingly diffuse, and eventually separates into two auditory images as the interaural correlation decreases. When the interaural delay increases from zero to several milliseconds, the auditory image of the binaurally identical noises also changes from a single image to two distinct images. However, measuring the effect of these two factors on an identical group of participants has not been investigated. This study examined the impacts of interaural correlation and delay on detecting a binaurally uncorrelated fragment (interaural correlation = 0) embedded in the binaurally correlated noises (i.e., binaural gap or break in interaural correlation). We found that the minimum duration of the binaural gap for its detection (i.e., duration threshold) increased exponentially as the interaural delay between the binaurally identical noises increased linearly from 0 to 8 ms. When no interaural delay was introduced, the duration threshold also increased exponentially as the interaural correlation of the binaurally correlated noises decreased linearly from 1 to 0.4. A linear relationship between the effect of interaural delay and that of interaural correlation was described for listeners participating in this study: a 1 ms increase in interaural delay appeared to correspond to a 0.07 decrease in interaural correlation specific to raising the duration threshold. Our results imply that a tradeoff may exist between the impacts of interaural correlation and interaural delay on the subjective representation of sounds delivered to two human ears.
doi:10.1371/journal.pone.0126342
PMCID: PMC4488353  PMID: 26125970
5.  Preservation of bone mass and structure in hibernating black bears (Ursus americanus) through elevated expression of anabolic genes 
Functional & integrative genomics  2012;12(2):357-365.
Physical inactivity reduces mechanical load on the skeleton, which leads to losses of bone mass and strength in non-hibernating mammalian species. Although bears are largely inactive during hibernation, they show no loss in bone mass and strength. To obtain insight into molecular mechanisms preventing disuse bone loss, we conducted a large-scale screen of transcriptional changes in trabecular bone comparing winter hibernating and summer non-hibernating black bears using a custom 12,800 probe cDNA microarray. A total of 241 genes were differentially expressed (P<0.01 and fold change >1.4) in the ilium bone of bears between winter and summer. The Gene Ontology and Gene Set Enrichment Analysis showed an elevated proportion in hibernating bears of overexpressed genes in six functional sets of genes involved in anabolic processes of tissue morphogenesis and development including skeletal development, cartilage development, and bone biosynthesis. Apoptosis genes demonstrated a tendency for downregulation during hibernation. No coordinated directional changes were detected for genes involved in bone resorption, although some genes responsible for osteoclast formation and differentiation (Ostf1, Rab9a, and c-Fos) were significantly underexpressed in bone of hibernating bears. Elevated expression of multiple anabolic genes without induction of bone resorption genes, and the down regulation of apoptosis-related genes, likely contribute to the adaptive mechanism that preserves bone mass and structure through prolonged periods of immobility during hibernation.
doi:10.1007/s10142-012-0266-3
PMCID: PMC4480364  PMID: 22351243
Hibernation; Bone biosynthesis; Gene expression; Apoptosis
6.  Study of celiac artery variations and related surgical techniques in gastric cancer 
AIM: To investigate celiac artery variations in gastric cancer patients and the impact on gastric cancer surgery, and also to discuss the value of the ultrasonic knife in reducing the risk caused by celiac artery variations.
METHODS: A retrospective analysis was conducted to investigate the difference in average operation time, intraoperative blood loss, number of harvested lymph nodes, average postoperative drainage within 3 d, and postoperative hospital stay between the group with vascular variations and no vascular variations, and between the ultrasonic harmonic scalpel and conventional electric scalpel surgery group.
RESULTS: One hundred and fifty-eight cases presented with normal celiac artery, and 80 presented with celiac artery variation (33.61%). The average operation time, blood loss, average drainage within 3 d after surgery in the celiac artery variation group were significantly more than in the no celiac artery variation group (215.7 ± 32.7 min vs 204.2 ± 31.3 min, 220.0 ± 56.7 mL vs 163.1 ± 52.3 mL, 193.6 ± 41.4 mL vs 175.3 ± 34.1 mL, respectively, P < 0.05). In celiac artery variation patients, the average operation time, blood loss, average drainage within 3 d after surgery in the ultrasonic harmonic scalpel group were significantly lower than in the conventional electric scalpel surgery group (209.5 ± 34.9 min vs 226.9 ± 29.4 min, 207.5 ± 57.1 mL vs 235.6 ± 52.9 mL, 184.4 ± 38.2 mL vs 205.0 ± 42.9 mL, respectively, P < 0.05), and the number of lymph node dissections was significantly higher than in the conventional surgery group (25.5 ± 9.2 vs 19.9 ± 7.8, P < 0.05).
CONCLUSION: Celiac artery variation increases the difficulty and risk of radical gastrectomy. Preoperative imaging evaluation and the application of ultrasonic harmonic scalpel are conducive to radical gastrectomy.
doi:10.3748/wjg.v21.i22.6944
PMCID: PMC4462736  PMID: 26078572
Celiac artery; Variation; MSCTA; Ultrasonic harmonic scalpel; Gastric cancer
7.  Archaeal TFEα/β is a hybrid of TFIIE and the RNA polymerase III subcomplex hRPC62/39 
eLife  null;4:e08378.
Transcription initiation of archaeal RNA polymerase (RNAP) and eukaryotic RNAPII is assisted by conserved basal transcription factors. The eukaryotic transcription factor TFIIE consists of α and β subunits. Here we have identified and characterised the function of the TFIIEβ homologue in archaea that on the primary sequence level is related to the RNAPIII subunit hRPC39. Both archaeal TFEβ and hRPC39 harbour a cubane 4Fe-4S cluster, which is crucial for heterodimerization of TFEα/β and its engagement with the RNAP clamp. TFEα/β stabilises the preinitiation complex, enhances DNA melting, and stimulates abortive and productive transcription. These activities are strictly dependent on the β subunit and the promoter sequence. Our results suggest that archaeal TFEα/β is likely to represent the evolutionary ancestor of TFIIE-like factors in extant eukaryotes.
DOI: http://dx.doi.org/10.7554/eLife.08378.001
eLife digest
Life on Earth is often categorized into three domains: the eukaryotes (which include plants, animals and fungi), the bacteria and a group of unusual, single-celled microorganisms called the archaea. But several recent discoveries suggest that the origin of the eukaryotes lies within the archaeal domain. The genetic material of all of these living organisms is made up of DNA, and genes within DNA contain the instructions to make other biological molecules. Making these molecules involves first copying these instructions into a molecule of RNA via a process called transcription.
All three domains of life use enzymes called RNA polymerases (RNAPs) for transcription, and all RNAPs are thought to have originated from a common ancestor. Archaea and bacteria have a single type of RNAP, whereas all eukaryotes have at least four different kinds of RNAP. The RNAPs found in archaea share many common features with their eukaryotic counterparts. In both cases, the RNAPs do not work alone. Instead, a class of proteins known as transcription factors assist in the first step of the transcription process. One of the eukarotyic RNAPs, termed RNAP II, works with a transcription factor that contains two protein subunits (called TFIIEα and TFIIEβ). While the archaeal counterpart for TFIIEα (called TFEα) is known, the counterpart for TFIIEβ is not.
Blombach et al. have now identified the archaeal counterpart of TFIIEβ in a species of archaea called Sulfolobus and have renamed it TFEβ. Sulfolobus cells are unable to survive without this protein, which works in a similar way to TFIIEβ in assisting the RNAP to start transcription. Further analyses show that the TFEβ protein is actually related to a protein subunit that is unique to RNAP III, another eukarotyic RNAP. Both of these proteins contain clusters of iron and sulphur. Blombach et al. also found that these iron-sulphur clusters enable TFEβ to bind to its TFEα partner to form a transcription factor that can interact with the RNAP and help it to carry out transcription.
These results suggest that the TFEα/β transcription factor found in archaea is likely to resemble the ancestor of the TFIIE transcription factors found in living eukaryotes. This discovery provides new insights in the evolutionary history of both the archaeal and the eukaryotic transcription machineries.
DOI: http://dx.doi.org/10.7554/eLife.08378.002
doi:10.7554/eLife.08378
PMCID: PMC4495717  PMID: 26067235
archaea; transcription; RNA polymerase; TFE; TFIIE; evolution; other
8.  CUL9 mediates the functions of the 3M complex and ubiquitylates survivin to maintain genome integrity 
Molecular cell  2014;54(5):805-819.
SUMMARY
The Cullin 9 (CUL9) gene encodes a putative E3 ligase that localizes in the cytoplasm. Cul9 null mice develop spontaneous tumors in multiple organs, however either the cellular or molecular mechanisms of CUL9 in tumor suppression are currently not known. We show here that deletion of Cul9 leads to abnormal nuclear morphology, increased DNA damage and aneuploidy. CUL9 knockdown rescues the microtubule and mitosis defects in cells depleted for CUL7 or OBSL1, two genes that are mutated in a mutually exclusive manner in 3M growth retardation syndrome and function in microtubule dynamics. CUL9 promotes the ubiquitylation and degradation of survivin and is inhibited by CUL7. Depletion of CUL7 decreases survivin level and overexpression of survivin rescues the defects caused by CUL7 depletion. We propose a 3M–CUL9-survivin pathway in maintaining microtubule and genome integrity, normal development and tumor suppression.
doi:10.1016/j.molcel.2014.03.046
PMCID: PMC4172312  PMID: 24793696
cullins; genome integrity; 3M disease; survivin
9.  The 3M complex maintains microtubule and genome integrity 
Molecular cell  2014;54(5):791-804.
SUMMARY
CUL7, OBSL1, and CCDC8 genes are mutated in a mutually exclusive manner in 3M and other growth retardation syndromes. The mechanism underlying the function of the three 3M genes in development is not known. We found that OBSL1 and CCDC8 form a complex with CUL7 and regulate the level and centrosomal localization of CUL7, respectively. CUL7 depletion results in altered microtubule dynamics, prometaphase arrest, tetraploidy and mitotic cell death. These defects are recaptured in CUL7 mutated 3M cells and can be rescued by wild-type, but not 3M patients-derived CUL7 mutants. Depletion of either OBSL1 or CCDC8 results in similar defects and sensitizes cells to microtubule damage as loss of CUL7 function. Microtubule damage reduces the level of CCDC8 that is required for the centrosomal localization of CUL7. We propose that CUL7, OBSL1, and CCDC8 proteins form a 3M complex that functions in maintaining microtubule and genome integrity and normal development.
doi:10.1016/j.molcel.2014.03.047
PMCID: PMC4165194  PMID: 24793695
10.  The Caenorhabditis elegans protein SAS-5 forms large oligomeric assemblies critical for centriole formation 
eLife  null;4:e07410.
Centrioles are microtubule-based organelles crucial for cell division, sensing and motility. In Caenorhabditis elegans, the onset of centriole formation requires notably the proteins SAS-5 and SAS-6, which have functional equivalents across eukaryotic evolution. Whereas the molecular architecture of SAS-6 and its role in initiating centriole formation are well understood, the mechanisms by which SAS-5 and its relatives function is unclear. Here, we combine biophysical and structural analysis to uncover the architecture of SAS-5 and examine its functional implications in vivo. Our work reveals that two distinct self-associating domains are necessary to form higher-order oligomers of SAS-5: a trimeric coiled coil and a novel globular dimeric Implico domain. Disruption of either domain leads to centriole duplication failure in worm embryos, indicating that large SAS-5 assemblies are necessary for function in vivo.
DOI: http://dx.doi.org/10.7554/eLife.07410.001
eLife digest
Most animal cells contain structures known as centrioles. Typically, a cell that is not dividing contains a pair of centrioles. But when a cell prepares to divide, the centrioles are duplicated. The two pairs of centrioles then organize the scaffolding that shares the genetic material equally between the newly formed cells at cell division.
Centriole assembly is tightly regulated and abnormalities in this process can lead to developmental defects and cancer. Centrioles likely contain several hundred proteins, but only a few of these are strictly needed for centriole assembly. New centrioles usually assemble from a cartwheel-like arrangement of proteins, which includes a protein called SAS-6. In the worm Caenorhabditis elegans, SAS-6 associates with another protein called SAS-5. This interaction is essential for centrioles to form, but the reason behind this is not clearly understood.
Now, Rogala et al. have used a range of techniques including X-ray crystallography, biophysics and studies of worm embryos to investigate the role of SAS-5 in C. elegans. These experiments revealed that SAS-5 proteins can interact with each other, via two regions of each protein termed a ‘coiled-coil’ and a previously unrecognized ‘Implico domain’. These regions drive the formation of assemblies that contain multiple SAS-5 proteins.
Next, Rogala et al. asked whether SAS-5 assemblies are important for centriole duplication. Mutant worm embryos, in which SAS-5 proteins could not interact with one another, failed to form new centrioles. This resulted in defects with cell division. An independent study by Cottee, Muschalik et al. obtained similar results and found that the fruit fly equivalent of SAS-5, called Ana2, can also self-associate and this activity is required for centriole duplication.
Further work is now needed to understand how SAS-5 and SAS-6 work with each other to form the initial framework at the core of centrioles.
DOI: http://dx.doi.org/10.7554/eLife.07410.002
doi:10.7554/eLife.07410
PMCID: PMC4471805  PMID: 26023830
centrioles; crystallography; structures; C. elegans
11.  Hydrogen sulfide is a novel gasotransmitter with pivotal role in regulating lateral root formation in plants 
Plant Signaling & Behavior  2014;9:e29127.
Hydrogen sulfide (H2S), the third gasotransmitter after nitric oxide (NO) and carbon monoxide (CO), is a critical neuromodulator in the pathogenesis of various diseases from neurodegenerative diseases to diabetes or heart failure. The crosstalk between NO and H2S has been well established in mammalian physiology. In planta, NO is demonstrated to regulate lateral root formation by acting downstream of auxin. The recent reports revealed that H2S is a novel inducer of lateral root (LR) formation by stimulating the expression of cell cycle regulatory genes (CCRGs), acting similarly with NO, CO, and IAA. Interestingly, during the initiation of lateral root primordia, IAA is a potent inducer of endogenous H2S and CO, which is produced by L-cysteine desulfhydrase (LCD) and heme oxygenase-1 (HO-1), respectively. The increasing evidences suggest that H2S-promoted LR growth is dependent on the endogenous production of CO. In addition, our results indicate that the H2S signaling in the regulation of LR formation can be associated to NO and Ca2+. In this addendum, we advanced a proposed schematic model for H2S-mediated signaling pathway of plant LR development.
doi:10.4161/psb.29127
PMCID: PMC4203638  PMID: 24832131
auxin; calcium; carbon monoxide; gasotransmitter; hydrogen sulfide; lateral root; signal molecule
12.  Safety of tirofiban for patients with acute ischemic stroke in routine clinical practice 
The aim of the present study was to investigate the safety of tirofiban alone and in combination with various treatments in acute ischemic stroke (AIS). A total of 120 patients with AIS were included in the study, and these patients were divided into three treatment groups: Group A (tirofiban alone, n=68), group B (tirofiban plus thrombolytic therapy, n=26), and group C (tirofiban as a ‘bridging therapy’, n=26). Risk factors, stroke severity, initial imaging, treatment regimens, complications and long-term outcomes were analyzed. In total, eight patients (6.7%) [six patients (23.1%) in group B and two patients (7.7%) in group C] had hemorrhage during or subsequent to treatment. Sixteen patients (six in group A, eight in group B and two in group C) succumbed during the hospital admission period. The mortality rate was 13.3% (8.8% for group A, 30.7% for group B and 7.7% for group C) in the acute phase. A favorable outcome (modified Rankin Scale score, 0–2) in the first three months after stroke was only observed in 43.3% of patients (44.1% in group A, 46.7% in group B and 36.4% in group C). The mean Barthel index was 72.3 in group A, 84.4 in group B and 56.8 in group C (total patient population, 71.0). The results of the present study have shown that stroke treatment with tirofiban is safe in AIS. A large randomized controlled trial in the future is required to decrease the incidence of the minor bleeding complications associated with tirofiban therapy.
doi:10.3892/etm.2015.2495
PMCID: PMC4486794  PMID: 26170930
tirofiban; acute ischemic stroke; safety
13.  Long-term oncologic outcomes of laparoscopic vs open surgery for stages II and III rectal cancer: A retrospective cohort study 
AIM: To evaluate the 5-year survival after laparoscopic surgery vs open surgery for stages II and III rectal cancer.
METHODS: This study enrolled 406 consecutive patients who underwent curative resection for stages II and III rectal cancer between January 2000 and December 2009 [laparoscopic rectal resection (LRR), n = 152; open rectal resection (ORR), n = 254]. Clinical characteristics, operative outcomes, pathological outcomes, postoperative recovery, and 5-year survival outcomes were compared between the two groups.
RESULTS: Most of the clinical characteristics were similar except age (59 years vs 55 years, P = 0.033) between the LRR group and ORR group. The proportion of anterior resection was higher in the LRR group than that in the ORR group (81.6% vs 66.1%, P = 0.001). The LRR group had less estimated blood loss (50 mL vs 200 mL, P < 0.001) and a lower rate of blood transfusion (4.6% vs 11.8%, P = 0.019) compared to the ORR group. The pathological outcomes of the two groups were comparable. The LRR group was associated with faster recovery of bowel function (2.8 d vs 3.7 d, P < 0.001) and shorter postoperative hospital stay (11.7 d vs 13.7 d, P < 0.001). The median follow-up time was 63 mo in the LRR group and 65 mo in the ORR group. As for the survival outcomes, the 5-year local recurrence rate (16.0% vs 16.4%, P = 0.753), 5-year disease-free survival (DFS) rate (63.0% vs 63.1%, P = 0.589), and 5-year overall survival (OS) rate (68.1% vs 63.5%, P = 0.682) were comparable between the LRR group and the ORR group. Stage by stage, there were also no statistical differences between the LRR group and the ORR group in terms of the 5-year local recurrence rate (stage II: 6.3% vs 8.7%, P = 0.623; stage III: 26.4% vs 23.2%, P = 0.747), 5-year DFS rate (stage II: 77.5% vs 77.6%, P = 0.462; stage III: 46.5% vs 50.9%, P = 0.738), and 5-year OS rate (stage II: 81.4% vs 74.3%, P = 0.242; stage III: 53.9% vs 54.1%, P = 0.459).
CONCLUSION: LRR for stages II and III rectal cancer can yield comparable long-term survival while achieving short-term benefits compared to open surgery.
doi:10.3748/wjg.v21.i18.5505
PMCID: PMC4427672  PMID: 25987773
Laparoscopic surgery; Locally advanced rectal cancer; Oncologic outcomes
14.  Pyruvate carboxylase is critical for non–small-cell lung cancer proliferation 
Anabolic biosynthesis requires precursors supplied by the Krebs cycle, which in turn requires anaplerosis to replenish precursor intermediates. The major anaplerotic sources are pyruvate and glutamine, which require the activity of pyruvate carboxylase (PC) and glutaminase 1 (GLS1), respectively. Due to their rapid proliferation, cancer cells have increased anabolic and energy demands; however, different cancer cell types exhibit differential requirements for PC- and GLS-mediated pathways for anaplerosis and cell proliferation. Here, we infused patients with early-stage non–small-cell lung cancer (NSCLC) with uniformly 13C-labeled glucose before tissue resection and determined that the cancerous tissues in these patients had enhanced PC activity. Freshly resected paired lung tissue slices cultured in 13C6-glucose or 13C5,15N2-glutamine tracers confirmed selective activation of PC over GLS in NSCLC. Compared with noncancerous tissues, PC expression was greatly enhanced in cancerous tissues, whereas GLS1 expression showed no trend. Moreover, immunohistochemical analysis of paired lung tissues showed PC overexpression in cancer cells rather than in stromal cells of tumor tissues. PC knockdown induced multinucleation, decreased cell proliferation and colony formation in human NSCLC cells, and reduced tumor growth in a mouse xenograft model. Growth inhibition was accompanied by perturbed Krebs cycle activity, inhibition of lipid and nucleotide biosynthesis, and altered glutathione homeostasis. These findings indicate that PC-mediated anaplerosis in early-stage NSCLC is required for tumor survival and proliferation.
doi:10.1172/JCI72873
PMCID: PMC4319441  PMID: 25607840
15.  Hedgehog signaling downregulates Suppressor of Fused through the HIB/SPOP-Crn axis in Drosophila 
Cell Research  2014;24(5):595-609.
Hedgehog (Hh) signaling plays vital roles in animal development and tissue homeostasis, and its misregulation causes congenital diseases and several types of cancer. Suppressor of Fused (Su(fu)) is a conserved inhibitory component of the Hh signaling pathway, but how it is regulated remains poorly understood. Here we demonstrate that in Drosophila Hh signaling promotes downregulation of Su(fu) through its target protein HIB (Hh-induced BTB protein). Interestingly, although HIB-mediated downregulation of Su(fu) depends on the E3 ubiquitin ligase Cul3, HIB does not directly regulate Su(fu) protein stability. Through an RNAi-based candidate gene screen, we identify the spliceosome factor Crooked neck (Crn) as a regulator of Su(fu) level. Epistasis analysis indicates that HIB downregulates Su(fu) through Crn. Furthermore, we provide evidence that HIB retains Crn in the nucleus, leading to reduced Su(fu) protein level. Finally, we show that SPOP, the mammalian homologue of HIB, can substitute HIB to downregulate Su(fu) level in Drosophila. Our study suggests that Hh regulates both Ci and Su(fu) levels through its target HIB, thus uncovering a novel feedback mechanism that regulates Hh signal transduction. The dual function of HIB may provide a buffering mechanism to fine-tune Hh pathway activity.
doi:10.1038/cr.2014.29
PMCID: PMC4011344  PMID: 24603360
Hh; Su(fu); HIB; Crn; SPOP; Cul3
16.  The miR-193a-3p-regulated ING5 gene activates the DNA damage response pathway and inhibits multi-chemoresistance in bladder cancer 
Oncotarget  2015;6(12):10195-10206.
As the major barrier to curative cancer chemotherapy, chemoresistance presents a formidable challenge to both cancer researchers and clinicians. We have previously shown that the bladder cancer (BCa) cell line 5637 is significantly more sensitive to the cytoxicity of five chemotherapeutic agents than H-bc cells. Using an RNA-seq-based omic analysis and validation at both the mRNA and protein levels, we found that the inhibitor of growth 5 (ING5) gene was upregulated in 5637 cells compared with H-bc cells, indicating that it has an inhibitory role in BCa chemoresistance. siRNA-mediated inhibition of ING5 increased the chemoresistance and inhibited the DNA damage response pathway in 5637 cells. Conversely, forced expression of EGFP-ING5 decreased the chemoresistance of and activated the DNA damage response pathway in H-bc cells. We also showed that ING5 gene expression is inhibited by miR-193a-3p and is instrumental in miR-193a-3p's role in activating BCa chemoresistance. Our results demonstrate both the role and mechanism of inhibition of BCa chemoresistance by ING5.
PMCID: PMC4496349  PMID: 25991669
ING5; miR-193a-3p; chemoresistance; bladder cancer; protein acetylation
17.  Intestinal mucus-derived nanoparticles mediate activation of Wnt/β-catenin signaling plays a role in induction of liver NKT cell anergy 
Hepatology (Baltimore, Md.)  2013;57(3):1250-1261.
The Wnt/β-catenin pathway has been known to play a role in induction of immune tolerance, but its role in the induction and maintenance of NKT cell anergy is unknown. We found that activation of the Wnt pathway(s) in the liver microenvironment is important for induction of NKT cell anergy. We identified a number of stimuli triggering Wnt/β-catenin pathway activation, including exogenous NKT cell activator, glycolipid α-GalCer, and endogenous PGE2. Glycolipid α-GalCer treatment of mice induced the expression of wnt3a and wnt5a in the liver, and subsequently resulted in a liver microenvironment that induced NKT cell anergy to α-GalCer restimulation. We also found that circulating PGE2 carried by nanoparticles is stable, and that these nanoparticles are A33+. A33+ is a marker of intestinal epithelial cells, which suggests that the nanoparticles are derived from the intestine. Mice treated with PGE2 associated with intestinal mucus-derived exosome-like nanoparticles (IDEN) induced NKT cell anergy. PGE2 treatment leads to activation of the Wnt/β-catenin pathway by inactivation of GSK-3β of NKT cells. IDEN-associated PGE2 also induces NKT cell anergy through modification of the ability of DCs to induce IL-12 and IFN-β in the context of both glycolipid presentation and TLR-mediated pathways. These findings demonstrate that IDEN associated PGE2 serves as an endogenous immune modulator between the liver and intestines and maintains liver NKT cell homeostasis and this finding has implications for development of NKT cell–based immunotherapies.
doi:10.1002/hep.26086
PMCID: PMC4414328  PMID: 22991247
Intestinal mucus-derived nanoparticles; Exosomes; Wnt and PGE2 signaling; liver NKT anergy
18.  Meta-analysis of acupuncture therapy for the treatment of stable angina pectoris 
Angina pectoris is a common symptom imperiling patients’ life quality. The aim of this study is to evaluate the efficacy and safety of acupuncture for stable angina pectoris. Clinical randomized-controlled trials (RCTs) comparing the efficacy of acupuncture to conventional drugs in patients with stable angina pectoris were searched using the following database of PubMed, Medline, Wanfang and CNKI. Overall odds ratio (ORs) and weighted mean difference (MD) with their 95% confidence intervals (CI) were calculated by using fixed- or random-effect models depending on the heterogeneity of the included trials. Total 8 RCTs, including 640 angina pectoris cases with 372 patients received acupuncture therapy and 268 patients received conventional drugs, were included. Overall, our result showed that acupuncture significantly increased the clinical curative effects in the relief of angina symptoms (OR=2.89, 95% CI=1.87-4.47, P<0.00001) and improved the electrocardiography (OR=1.83, 95% CI=1.23-2.71, P=0.003), indicating that acupuncture therapy was superior to conventional drugs. Although there was no significant difference in overall effective rate relating reduction of nitroglycerin between two groups (OR=2.13, 95% CI=0.90-5.07, P=0.09), a significant reduction on nitroglycerin consumption in acupuncture group was found (MD=-0.44, 95% CI=-0.64, -0.24, P<0.0001). Furthermore, the time to onset of angina relief was longer for acupuncture therapy than for traditional medicines (MD=2.44, 95% CI=1.64-3.24, P<0.00001, min). No adverse effects associated with acupuncture therapy were found. Acupuncture may be an effective therapy for stable angina pectoris. More clinical trials are needed to systematically assess the role of acupuncture in angina pectoris.
PMCID: PMC4483941  PMID: 26131084
Stable angina pectoris; acupuncture; meta-analysis
19.  Delivery of therapeutic agents by nanoparticles made of grapefruit-derived lipids 
Nature communications  2013;4:1867.
Although the use of nanotechnology for the delivery of a wide range of medical treatments has potential to reduce adverse effects associated with drug therapy, tissue-specific delivery remains challenging. Here we show that nanoparticles made of grapefruit-derived lipids, which we call grapefruit-derived nanovectors (GNVs), can transport chemotherapeutic agents, siRNA, DNA expression vectors and proteins to different types of cells. We demonstrate the in vivo targeting specificity of GNVs by co-delivering therapeutic agents with folic acid, which in turn leads to significantly increasing targeting efficiency to cells expressing folate receptors. The therapeutic potential of GNVs was further demonstrated by enhancing the chemotherapeutic inhibition of tumor growth in two tumor animal models. GNVs are less toxic than nanoparticles made of synthetic lipids and, when injected intravenously into pregnant mice, do not pass the placental barrier, suggesting they may be a useful tool for drug delivery.
doi:10.1038/ncomms2886
PMCID: PMC4396627  PMID: 23695661
20.  Exosomes-like nanoparticles from intestinal mucosal cells carry prostaglandin E2 and suppress activation of liver natural killer T cells 
Regulation and induction of anergy in natural killer T (NKT) cells of the liver can inhibit autoimmune and anti-tumor responses by mechanisms that are poorly understood. We investigated the effects of prostaglandin E2 (PGE2), delivered by intestinal, mucus-derived, exosome-like nanoparticles (IDENs), on NKT cells in mice. Here, we demonstrate that IDENs migrate to the liver where they induce NKT cell anergy. These effects were mediated by an IDENs PGE2. Blocking PGE2 synthesis attenuated IDENs inhibition of induction of IFN-γ and IL-4 by α-GalCer stimulated liver NKT cells in a PGE2 EP2/EP4 receptor mediated manner. Pro-inflammatory conditions enhanced the migration of IDENs to the liver where α-GalCer and PGE2 induced NKT anergy in response to subsequent α-GalCer stimulation. These findings demonstrate that IDENs carrying PGE2 can be transferred from the intestine to the liver, where they act as immune modulators, inducing an anergic-like state of NKT cells. These reagents might be developed as therapeutics for autoimmune liver diseases.
doi:10.4049/jimmunol.1203170
PMCID: PMC4396634  PMID: 23467936
liver disease; mouse model; conA; immune regulation; IL-4
21.  δ-Tocotrienol Induces Human Bladder Cancer Cell Growth Arrest, Apoptosis and Chemosensitization through Inhibition of STAT3 Pathway 
PLoS ONE  2015;10(4):e0122712.
Vitamin E intake has been implicated in reduction of bladder cancer risk. However, the mechanisms remain elusive. Here we reported that δ-tocotrienol (δ-T3), one of vitamin E isomers, possessed the most potent cytotoxic capacity against human bladder cancer cells, compared with other Vitamin E isomers. δ-T3 inhibited cancer cell proliferation and colonogenicity through induction of G1 phase arrest and apoptosis. Western blotting assay revealed that δ-T3 increased the expression levels of cell cycle inhibitors (p21, p27), pro-apoptotic protein (Bax) and suppressed expression levels of cell cycle protein (Cyclin D1), anti-apoptotic proteins (Bcl-2, Bcl-xL and Mcl-1), resulting in the Caspase-3 activation and cleavage of PARP. Moreover, the δ-T3 treatment inhibited ETK phosphorylation level and induced SHP-1 expression, which was correlated with downregulation of STAT3 activation. In line with this, δ-T3 reduced the STAT3 protein level in nuclear fraction, as well as its transcription activity. Knockdown of SHP-1 partially reversed δ-T3-induced cell growth arrest. Importantly, low dose of δ-T3 sensitized Gemcitabine-induced cytotoxic effects on human bladder cancer cells. Overall, our findings demonstrated, for the first time, the cytotoxic effects of δ-T3 on bladder cancer cells and suggest that δ-T3 might be a promising chemosensitization reagent for Gemcitabine in bladder cancer treatment.
doi:10.1371/journal.pone.0122712
PMCID: PMC4388509  PMID: 25849286
22.  Puerarin exhibits greater distribution and longer retention time in neurons than astrocytes in a co-cultured system 
Neural Regeneration Research  2015;10(4):605-609.
The phytoestrogen puerarin has been shown to protect neurons and astrocytes in the brain, and is therefore an attractive drug in the treatment of Alzheimer's disease, Parkinson's disease and cerebral ischemia. Whether puerarin exhibits the same biological processes in neurons and astrocytes in vitro has rarely been reported. In this study, cortical neurons and astrocytes of newborn Sprague-Dawley rats were separated, identified and co-cultured in a system based on Transwell membranes. The retention time and distribution of puerarin in each cell type was detected by fluorescence spectrophotometry and fluorescence microscope. The concentration of puerarin in both co-cultured and separately cultured neurons was greater than that of astrocytes. Puerarin concentration reached a maximum 20 minutes after it was added. At 60 minutes after its addition, a scant amount of drug was detected in astrocytes; however in both separately cultured and co-cultured neurons, the concentration of puerarin achieved a stable level of about 12.8 ng/mL. The results indicate that puerarin had a higher concentration and longer retention time in neurons than that observed in astrocytes.
doi:10.4103/1673-5374.155435
PMCID: PMC4424754  PMID: 26170822
nerve regeneration; puerarin; in vitro experiments; co-culture; neurons; astrocytes; Transwell; neonatal rats; neural regeneration
23.  Family influences on physical activity and sedentary behaviours in Chinese junior high school students: a cross-sectional study 
BMC Public Health  2015;15:287.
Background
Family influence plays an important role in a child’s physical activity (PA). This study aimed to describe the level of moderate to vigorous intensity physical activity (MVPA) and sedentary behaviours among Chinese junior high school students and examine the associations between different types of family influence and MVPA or sedentary behaviours.
Methods
Participants of two independent cross-sectional surveys, conducted in 2009 and 2011, were students in Grade 7 and 9 from all junior high schools in Hangzhou, China. The daily duration and frequency of MVPA, amount of sedentary time and frequency of family support were self-reported. Multi-level mixed-effects logistic regression was used to examine the associations between different types or levels of family influence and MVPA or sedentary behaviours.
Results
A total of 7286 students were analysed finally. Overall, only 9.0% of the students participated in MVPA at least 60 minutes/day; 63.9% spent no more than 2 hours/day in sedentary behaviours. Frequent verbal encouragement and watching were associated with less leisure-time sedentary behaviours. The multivariate-adjusted odds ratios (ORs) for verbal encouragement and watching were 1.29 (95% CI, 1.08 to 1.55) and 1.19 (95% CI, 0.97 to 1.45) for 5-7 days per week. The involvement of family in the children’s activity in most days of the week was associated with both higher level of MVPA and less leisure-time sedentary behaviours. The respective ORs among students who reported familial support 5-7 days per week, were 1.50 (95% CI, 1.21 to 1.86) for engaging in seven days of MVPA per week, 1.67 (95% CI, 1.19 to 2.32) for at least 60 minutes of MVPA daily, and 1.48 (95% CI, 1.19 to 1.84) for no more than 2 hours of leisure-time sedentary behaviours daily.
Conclusions
This study found that less than 10.0% of urban Chinese adolescents engaged in MVPA at least 60 minutes/day. Family involving themselves in the children’s activity exerted the most significant influence on children’s behaviours as compared with verbally encouraging and observing children’s activity. Any type of familial support including verbally encouraging, watching, and involving had effects on reducing leisure-time sedentary behaviours.
Electronic supplementary material
The online version of this article (doi:10.1186/s12889-015-1593-9) contains supplementary material, which is available to authorized users.
doi:10.1186/s12889-015-1593-9
PMCID: PMC4376336  PMID: 25884212
Moderate to vigorous intensity; Physical activity; Sedentary; Family influence; Adolescents
24.  Dihydroartemisinin Inhibits Glucose Uptake and Cooperates with Glycolysis Inhibitor to Induce Apoptosis in Non-Small Cell Lung Carcinoma Cells 
PLoS ONE  2015;10(3):e0120426.
Despite recent advances in the therapy of non-small cell lung cancer (NSCLC), the chemotherapy efficacy against NSCLC is still unsatisfactory. Previous studies show the herbal antimalarial drug dihydroartemisinin (DHA) displays cytotoxic to multiple human tumors. Here, we showed that DHA decreased cell viability and colony formation, induced apoptosis in A549 and PC-9 cells. Additionally, we first revealed DHA inhibited glucose uptake in NSCLC cells. Moreover, glycolytic metabolism was attenuated by DHA, including inhibition of ATP and lactate production. Consequently, we demonstrated that the phosphorylated forms of both S6 ribosomal protein and mechanistic target of rapamycin (mTOR), and GLUT1 levels were abrogated by DHA treatment in NSCLC cells. Furthermore, the upregulation of mTOR activation by high expressed Rheb increased the level of glycolytic metabolism and cell viability inhibited by DHA. These results suggested that DHA-suppressed glycolytic metabolism might be associated with mTOR activation and GLUT1 expression. Besides, we showed GLUT1 overexpression significantly attenuated DHA-triggered NSCLC cells apoptosis. Notably, DHA synergized with 2-Deoxy-D-glucose (2DG, a glycolysis inhibitor) to reduce cell viability and increase cell apoptosis in A549 and PC-9 cells. However, the combination of the two compounds displayed minimal toxicity to WI-38 cells, a normal lung fibroblast cell line. More importantly, 2DG synergistically potentiated DHA-induced activation of caspase-9, -8 and -3, as well as the levels of both cytochrome c and AIF of cytoplasm. However, 2DG failed to increase the reactive oxygen species (ROS) levels elicited by DHA. Overall, the data shown above indicated DHA plus 2DG induced apoptosis was involved in both extrinsic and intrinsic apoptosis pathways in NSCLC cells.
doi:10.1371/journal.pone.0120426
PMCID: PMC4370589  PMID: 25799586
25.  Sensitization of P2X3 receptors by cystathionine β-synthetase mediates persistent pain hypersensitivity in a rat model of lumbar disc herniation 
Molecular Pain  2015;11:15.
Lumbar disc herniation (LDH) is a major cause of discogenic low back pain and sciatica, but the underlying mechanisms remain largely unknown. Hydrogen sulfide (H2S) is becoming recognized for its involvement in a wide variety of processes including inflammation and nociception. The present study was designed to investigate the roles of the H2S signaling pathway in the regulation of expression and function of purinergic receptors (P2XRs) in dorsal root ganglion (DRG) neurons from rats with LDH. LDH was induced by implantation of autologous nucleus pulposus (NP), harvested from rat tail, in lumbar 5 and 6 spinal nerve roots. Implantation of autologous NP induced persistent pain hypersensitivity, which was partially reversed by an intrathecal injection of A317491, a potent inhibitor of P2X3Rs and P2X2/3Rs. The NP induced persistent pain hypersensitivity was associated with the increased expression of P2X3Rs, but not P2X1Rs and P2X2Rs, receptors in L5-6 DRGs. NP implantation also produced a 2-fold increase in ATP-induced intracellular calcium signals in DRG neurons when compared to those of controls (P < 0.05). Interestingly, NP implantation significantly enhanced expression of the endogenous hydrogen sulfide producing enzyme, cystathionine-β-synthetase (CBS). Systematic administration of O-(Carboxymethyl) hydroxylamine hemihydrochloride (AOAA), an inhibitor of CBS, suppressed the upregulation of P2X3R expression and the potentiation of ATP-induced intracellular calcium signals in DRG neurons (P < 0.05). Intrathecal injection of AOAA markedly attenuated NP induced- persistent pain hypersensitivity. Our results suggest that sensitization of P2X3Rs, which is likely mediated by CBS-H2S signaling in primary sensory neurons, contributes to discogenic pain. Targeting CBS/H2S-P2X3R signaling may represent a potential treatment for neuropathic pain caused by LDH.
doi:10.1186/s12990-015-0012-7
PMCID: PMC4372268  PMID: 25885215
Lumbar disc herniation; Dorsal root ganglion; Neuropathic pain; Hydrogen sulfide; P2X receptors

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