Dichloroacetate induces protective autophagy in LoVo cells: involvement of cathepsin D/thioredoxin-like protein 1 and Akt-mTOR-mediated signaling

Dichloroacetate (DCA) is an inhibitor of pyruvate dehydrogenase kinase (PDK), and recently it has been shown as a promising nontoxic antineoplastic agent. In this study, we demonstrated that DCA could induce autophagy in LoVo cells, which were confirmed by the formation of autophagosomes, appearance of punctate patterns of LC3 immunoreactivity and activation of autophagy associated proteins. Moreover, autophagy inhibition by 3-methyladenine (3-MA) or Atg7 siRNA treatment can significantly enhance DCA-induced apoptosis. To determine the underlying mechanism of DCA-induced autophagy, target identification using drug affinity responsive target stability (DARTS) coupled with ESI-Q-TOF MS/MS analysis were utilized to profile differentially expressed proteins between control and DCA-treated LoVo cells. As a result, Cathepsin D (CTSD) and thioredoxin-like protein 1 (TXNL1) were identified with significant alterations compared with control. Further study indicated that DCA treatment significantly promoted abnormal reactive oxygen species (ROS) production. On the other hand, DCA-triggered autophagy could be attenuated by N-acetyl cysteine (NAC), a ROS inhibitor. Finally, we demonstrated that the Akt-mTOR signaling pathway, a major negative regulator of autophagy, was suppressed by DCA treatment. To our knowledge, it was the first study to show that DCA induced protective autophagy in LoVo cells, and the potential mechanisms were involved in ROS imbalance and Akt-mTOR signaling pathway suppression.     

Proteomic analysis of mismatch repair-mediated alkylating agent-induced DNA damage response

Background

Mediating DNA damage-induced apoptosis is an important genome-maintenance function of the mismatch repair (MMR) system. Defects in MMR not only cause carcinogenesis, but also render cancer cells highly resistant to chemotherapeutics, including alkylating agents. To understand the mechanisms of MMR-mediated apoptosis and MMR-deficiency-caused drug resistance, we analyze a model alkylating agent (N-methyl-N’-nitro-N-nitrosoguanidine, MNNG)-induced changes in protein phosphorylation and abundance in two cell lines, the MMR-proficient TK6 and its derivative MMR-deficient MT1.

Results

Under an experimental condition that MNNG-induced apoptosis was only observed in MutSα-proficient (TK6), but not in MutSα-deficient (MT1) cells, quantitative analysis of the proteomic data revealed differential expression and phosphorylation of numerous individual proteins and clusters of protein kinase substrates, as well differential activation of response pathways/networks in MNNG-treated TK6 and MT1 cells. Many alterations in TK6 cells are in favor of turning on the apoptotic machinery, while many of those in MT1 cells are to promote cell proliferation and anti-apoptosis.

Conclusions

Our work provides novel molecular insights into the mechanism of MMR-mediated DNA damage-induced apoptosis.

     

Patulin induces pro-survival functions via autophagy inhibition and p62 accumulation

Patulin (PAT) is one of the most common mycotoxins found in moldy fruits. Skin contact is one of the most likely exposure routes of PAT. Investigation of dermal toxicity of PAT is clearly needed and has been highlighted by WHO. In the present study, using human keratinocyte HaCaT cells as a model, we found that treatment with PAT caused an increased autophagosome accumulation. Measurements of autophagic flux demonstrated that the accumulation of autophagosomes by PAT was not directly due to enhanced autophagosome formation but due to inhibition of autophagosome degradation. Reductions in the activities of the lysosomal enzymes cathepsin B and cathepsin D by PAT might contribute to this inhibitory effect. Consistent with this, inhibition of autophagosome degradation by PAT resulted in accumulation of p62 that functioned as a pro-survival signal. The pro-survival function of p62 was found to be attributed to reactive oxygen species-mediated cytoprotective endoplasmic reticulum (ER) stress response. ER stress exerted cytoprotective effect via extracellular signal-regulated kinase1/2-dependent B-cell CLL/lymphoma 2-associated agonist of cell death inhibitory phosphorylation. Given the critical role of autophagy and its substrate p62 in carcinogenesis, our findings may have important implications in PAT-induced skin carcinogenesis.     

Activation of TLR4 signaling promotes gastric cancer progression by inducing mitochondrial ROS production

Chronic infection, such as Helicobacter pylori infection, has been associated with the development of gastric cancer (GC). Pathogen-associated molecular patterns can trigger inflammatory responses via Toll-like receptors (TLRs) in GC. Here we showed that Toll-like receptor 4 (TLR4) was highly expressed in GC cells and was associated with the aggressiveness of GC. The binding of lipopolysaccharide (LPS) to TLR4 on GC cells enhanced proliferation without affecting apoptosis. Higher level of reactive oxygen species (ROS) was induced after activation of TLR4 signaling in GC. Using oxidase inhibitors and antioxidants, we found that mitochondrial ROS (mROS) was major source of TLR4-stimulated ROS generation. This elevated mROS production can be inhibited by diphenylene iodonium (DPI), and the blocking of the mROS production rather than ROS neutralization resulted in cell cycle arrest and the loss of mitochondrial potential, which were plausible reason for decreased cell viability. Furthermore, the increased mROS owing to TLR4 signaling resulted in the activation of Akt phosphorylation and NF-κB p65 nuclear translocation. Altogether, these results reveal a novel pathway linking innate immune signaling to GC cell proliferation, implicate mROS as an important component of cell survival signals and further establish mitochondria as hubs for GC therapies.     

USP7 inhibitor P22077 inhibits neuroblastoma growth via inducing p53-mediated apoptosis

Neuroblastoma (NB) is a common pediatric cancer and contributes to more than 15% of all pediatric cancer-related deaths. Unlike adult tumors, recurrent somatic mutations in NB, such as tumor protein 53 (p53) mutations, occur with relative paucity. In addition, p53 downstream function is intact in NB cells with wild-type p53, suggesting that reactivation of p53 may be a viable therapeutic strategy for NB treatment. Herein, we report that the ubiquitin-specific protease 7 (USP7) inhibitor, {"type":"entrez-protein","attrs":{"text":"P22077","term_id":"134707","term_text":"P22077"}}P22077, potently induces apoptosis in NB cells with an intact USP7-HDM2-p53 axis but not in NB cells with mutant p53 or without human homolog of MDM2 (HDM2) expression. In this study, we found that {"type":"entrez-protein","attrs":{"text":"P22077","term_id":"134707","term_text":"P22077"}}P22077 stabilized p53 by inducing HDM2 protein degradation in NB cells. {"type":"entrez-protein","attrs":{"text":"P22077","term_id":"134707","term_text":"P22077"}}P22077 also significantly augmented the cytotoxic effects of doxorubicin (Dox) and etoposide (VP-16) in NB cells with an intact USP7-HDM2-p53 axis. Moreover, {"type":"entrez-protein","attrs":{"text":"P22077","term_id":"134707","term_text":"P22077"}}P22077 was found to be able to sensitize chemoresistant LA-N-6 NB cells to chemotherapy. In an in vivo orthotopic NB mouse model, {"type":"entrez-protein","attrs":{"text":"P22077","term_id":"134707","term_text":"P22077"}}P22077 significantly inhibited the xenograft growth of three NB cell lines. Database analysis of NB patients shows that high expression of USP7 significantly predicts poor outcomes. Together, our data strongly suggest that targeting USP7 is a novel concept in the treatment of NB. USP7-specific inhibitors like {"type":"entrez-protein","attrs":{"text":"P22077","term_id":"134707","term_text":"P22077"}}P22077 may serve not only as a stand-alone therapy but also as an effective adjunct to current chemotherapeutic regimens for treating NB with an intact USP7-HDM2-p53 axis.     

Effects of fish oil on lymphocyte proliferation,cytokine production and intracellular signalling in weanling pigs

It has been widely documented that fish oil attenuates inflammatory responses partially via down-regulation of T-lymphocyte function. To determine the anti-inflammatory role of fish oil in weanling pigs, we investigated the effects of fish oil and its functional constituents on peripheral blood lymphocyte proliferation, cytokine production and subsequent intracellular signalling in inflammatory-challenged weanling pigs and in in vitro cultured lymphocytes. Fish oil (7%) or corn oil (7%) was supplemented to 72 crossbred pigs (7.6 ± 0.3 kg BW and 28 ± 3 days of age) in a 2 × 2 factorial experiment that included an Escherichia coli lipopolysaccharide (LPS) challenge (challenged or not challenged). On day 14 and 28 of the experiment, 200 μg/kg BW of LPS or an equivalent amount of sterile saline was administered to the pigs by intraperitoneal injection. Blood samples were collected on days 15 and 29 to determine peripheral blood lymphocyte proliferation, interleukin-1β (IL-1β) and interleukin-2 (IL-2) production. The results showed that inflammatory challenge decreased average daily gain (P < 0.05) and average daily feed intake (P < 0.05) during days 15 - 28. Fish oil supplementation had no effect on growth performance. Inflammatory challenge increased lymphocyte proliferative response to concanavalin A (Con A) (P < 0.05) following each challenge. Fish oil tended to suppress (P < 0.1) the proliferation following the first challenge. Similarly, fish oil tended to reduce IL-1β production (P < 0.1) following the second challenge and IL-2 (P < 0.1) production following the first challenge in both challenged and unchallenged pigs compared with corn oil. In parallel in vitro experiments, peripheral blood lymphocytes of weanling pigs were incubated with various concentrations of eicosapentaenoic acid (EPA), docosahexaenoic acid (DHA) or linoleic acid (LA) (0, 20, 40, 60, 80, 100 μg/ml). EPA, DHA and high levels of LA predominantly suppressed IL-1β (P < 0.05), IL-2 (P < 0.05) production and subsequent lymphocyte proliferation (P < 0.05). Low levels of LA increased (P < 0.05) IL-2 production. Compared with LA, EPA resulted in a stronger inhibition of lymphocyte proliferation (P < 0.05) and IL-2 (P < 0.01), and DHA resulted in a stronger inhibition of IL-1β (P < 0.05) and IL-2 (P < 0.01). To elucidate the mechanism(s) by which fish oil and its functional constituents suppressed lymphocyte function, the kinetics of intracellular [Ca^{2 +}]_i and protein kinase C activity were determined in in vitro experiments. EPA, DHA and LA exerted very similar dose-dependent stimulatory effects on intracellular Ca^{2 +}. EPA and DHA inhibited protein kinase C activity (P < 0.05), while LA had no significant effect (P > 0.05). These results suggest that fish oil and its functional constituents (EPA and DHA) exerted an anti-inflammatory effect by down-regulation of lymphocyte activation in weanling pigs, possibly by manipulation of intracellular signalling.     

The suitability of a faecal suspension of sheep as inocula for the estimation of utilizable crude protein of feeds by in vitro incubation

The objective of this review is to outline those parts of modelling approaches in pig production which are not highly developed; these are the partitioning of protein and lipid accretion in different anatomical body parts. The authors introduce present models with a critical evaluation and draw some conlusions for further developments. Based on present knowledge this paper demonstrates the process of protein and fat accretion in different body compartments in pigs and influencing factors. A further aim is to assist in the conceptual development of a new pig model, which is more detailed, precise and accurate than currently available models. Exsisting models are generally deficient with regard to the translation of lipid and protein gain into lean and fatty tissue. Only assumed values for this translation have been used so far and the concepts underlying these values are not well understood. Therefore, it may be appropriate to develop a compartimental model to predict protein and fat deposition in growing and fattening pigs. With this new approach the model can supply sufficiently the changing consumer demands regarding to the possibility of meat quality prediction.     

A multi-step directional generalized gradient vector flow snake for target tumor segmentation in US-guided high-intensity focused ultrasound ablation

Getting precise locations of target tumors can help to ensure ablation of cancerous tissues and avoid unwanted destruction of healthy tissues in high-intensity focused ultrasound (HIFU) treatment system. Because of speckle noise and spurious boundaries in ultrasound images, traditional image segmentation methods are not suitable for achieving the precise locations of target tumors in HIFU ablation. In this paper, a multi-step directional generalized gradient vector flow snake model is introduced for target tumor segmentation. In the first step, the traditional generalized gradient vector flow (GGVF) snake is used to obtain an approximate contour of the tumor. According to the approximate contour, a new distance map is generated. Subsequently, a new directional edge map is created by calculating a scalar product of the gradients of the distance map and the initial image. In this process, the gradient directional information and the magnitude information of the distance map are used to attenuate unwanted edges and highlight the real edges in the new directional edge map. Finally, a refined GGVF field is derived from a diffusion operation of the gradient vectors of the directional edge map. The GGVF field is used to refine the tumor's contour, by directing the approximate contour to edges with the desired gradient directionality. Based on the newly developed snake model, the influences of the spurious boundaries and the speckle noise are significantly reduced in the ultrasound image segmentation. Experimental results indicate that this technique is greatly useful for target tumor segmentation in HIFU treatment system     

泌尿生殖道非淋菌性感染的病原学以及耐药性分析

目的：探究泌尿生殖道非淋茵性感染的病原学以及耐药性,指导临床合理用药。方法：对我院2006年3月．2011年12月确诊的2136例非淋菌性泌尿生殖道感染患者进行标本采集,对病原体及其耐药性进行检测分析。结果：淋茵感染泌尿生殖道病原茵主要为CT及支原体,男性患者CT感染率高于女性患者,女性患者支原体感染率高于男性患者,此外,念珠茵、滴虫及其他难以检出细菌亦属于致病病原茵的一种;在12种抗生素中,多西环素、米诺环素及交沙霉素敏感率较高,均〉80％,罗红霉素、红霉素及环丙沙星敏感度较低,均〈20％。结论：非淋茵感染泌尿生殖道病原菌主要为CT及支原体,男性患者CT感染率高于女性患者,女性患者支原体感染率高于男性患者,病原茵对多西环素、米诺环素及交沙霉素耐药性较低,但不同地域病原茵耐药性亦存在差异,应按照实际情况进行耐药性检测,指导临床合理用药。