Leptin Enhances Hepatic Fibrosis and Inflammation in a Mouse Model of Cholestasis.

Leptin is an adipokine with roles in food intake and energy metabolism through its actions on neurons in the hypothalamus. The role of leptin in obesity and cardiovascular disorders is well documented. However, its influence on liver conditions such as cholestasis is poorly understood. The effects of exogenous leptin and leptin-neutralizing antibody on biliary hyperplasia, hepatic fibrosis, and inflammation in the multidrug resistance protein 2 knockout (Mdr2KO) mouse model of cholestasis were assessed by quantifying markers specific for cholangiocytes, activated hepatic stellate cells (HSCs), and cytokines. Serum and hepatic leptin were increased in Mdr2KO mice compared with FVB/NJ (FVBN) controls, and exogenous leptin enhanced biliary hyperplasia and liver fibrosis in Mdr2KO and FVBN mice. Leptin administration increased hepatic expression of C-C motif chemokine ligand 2 and IL-6 in Mdr2KO mice. In contrast, leptin-neutralizing antibody reduced intrahepatic bile duct mass and decreased HSC activation in Mdr2KO mice compared with FVBN controls. Sex-related differences were noted, with female Mdr2KO mice having more leptin than males. In cholangiocytes and LX2 cells in vitro, leptin increased phosphorylated Akt and stimulated cell proliferation. Leptin receptor siRNA and inhibitors of Akt phosphorylation impaired leptin-induced cell proliferation and proinflammatory cytokines. The current data suggest that leptin is abnormally increased in cholestatic mice, and excess leptin increases ductular reaction, hepatic fibrosis, and inflammation via leptin receptor-mediated phosphorylation of Akt in cholangiocytes and HSCs.

IRS4 promotes the progression of non-small cell lung cancer and confers resistance to EGFR-TKI through the activation of PI3K/Akt and Ras-MAPK pathways.

IRS4 is a member of the insulin receptor substrate (IRS) protein family. It acts as a cytoplasmic adaptor protein, integrating and transmitting signals from receptor protein tyrosine kinases to the intracellular environment. IRS4 can induce mammary tumorigenesis and is usually overexpressed in non-small cell lung cancer (NSCLC). However, little is known about the role of IRS4 in the development and progression of lung cancer. In this study, we show that IRS4 knockout suppresses the proliferation, colony formation, migration, and invasion of A549 lung cancer cells, as well as tumor growth in a nude mouse xenograft model. In contrast, stable expression of IRS4 showed the opposite effects. As expected, IRS4 was found to activate the PI3K/Akt and Ras-MAPK pathways, and we also showed that IRS4 depletion significantly enhanced the sensitivity of EGFR tyrosine kinase inhibitor (EGFR-TKI)-resistant cells to gefitinib. Taken together, these results show that IRS4 promotes NSCLC progression and may represent a potential therapeutic target for EGFR-TKI-resistant NSCLC.

Akt scaffold proteins: the key to controlling specificity of Akt signaling.

The phosphatidylinositol 3-kinase-Akt signaling pathway plays an essential role in regulating cell proliferation and apoptosis. Akt kinase is at the center of this signaling pathway and interacts with a variety of proteins. Akt is overexpressed in almost 80% of tumors. However, inhibiting Akt has serious clinical side effects so is not a suitable treatment for cancer. During recent years, Akt scaffold proteins have received increasing attention for their ability to regulate Akt signaling and have emerged as potential targets for cancer therapy. In this paper, we categorize Akt kinase scaffold proteins into four groups based on their cellular location: membrane-bound activator and inhibitor, cytoplasm, and endosome. We describe how these scaffolds interact with Akt kinase, how they affect Akt activity, and how they regulate the specificity of Akt signaling. We also discuss the clinical application of Akt scaffold proteins as targets for cancer therapy.

HDAC1-Smad3-mSin3A complex is required for Smad3-induced transcriptional inhibition of hepatocyte growth factor receptor in human lung cancers.

c-Met hyperactivity has been observed in numerous neoplasms. Several researchers have shown that the abnormal activation of c-Met is mainly caused by transcriptional activation. However, the molecular mechanism behind this transcriptional regulation is poorly understood. Here, we suggest that Smad3 negatively regulates the expression and activation of c-Met via a transcriptional mechanism. We explore the molecular mechanisms that underlie Smad3-induced c-Met transcription inhibition. We found in contrast to the high expression of c-Met, Smad3 showed low protein and mRNA levels. Smad3 and c-Met expressions were inconsistent between lung cancer tissues and cell lines. We also found that Smad3 overexpression suppresses whereas Smad3 knockdown significantly promotes Epithelial-Mesenchymal Transition and production of the angiogenic factors VEGF, CTGF and COX-2 through the ERK1/2 pathway. In addition, Smad3 overexpression decreases whereas Smad3 knockdown significantly increases protein and mRNA levels of invasion-related ß-catenin and FAK through the PI3K/Akt pathway. Furthermore, using the chromatin immunoprecipitation analysis method, we demonstrate that a transcriptional regulatory complex consisting of HDAC1, Smad3 and mSin3A binds to the promoter of the c-Met gene. By either silencing endogenous mSin3A expression with siRNA or by pretreating cells with a specific HDAC1 inhibitor (MS-275), Smad3-induced transcriptional suppression of c-Met could be effectively attenuated. These results demonstrate that Smad3-induced inhibition of c-Met transcription depends on of a functional transcriptional regulatory complex that includes Smad3, mSin3A and HDAC1 at the c-Met promoter. Collectively, our findings reveal a new regulatory mechanism of c-Met signaling, and suggest a potential molecular target for the development of anticancer drugs.

The C-terminal of the α1b-adreneroceptor is a key determinant for its structure integrity and biological functions.

The C-terminal of G protein-coupled receptors is now recognized as being important for G protein activation and signaling function. To detect the role of C-terminal tail in receptor activation, we used the α1b-AR, which has a long C-terminal of 164 amino acids. We constructed the intramolecular FRET sensors, in which the C-terminal was truncated to 10 (∆C-10), 20 (∆C-20), 30 (∆C-30), 50 (∆C-50), 70 (∆C-70), or 90 (∆C-90). The truncated mutants of ∆C-10, ∆C-20, or ∆C-30 cannot induce FRET signal changes and downstream ERK1/2 phosphorylation. However, the truncated mutants of ∆C-50, ∆C-70, or ∆C-90 induce significant FRET signal changes and downstream ERK1/2 phosphorylation, especially ∆C-90. This is particularly true in the case of the ∆C-90, ∆C-70, or ∆C-50 which retained the potential phosphorylation sites (Ser401, Ser404, Ser408, or Ser410). The ∆C-90 showed an increase in agonist-induced FRET signal changes and ERK1/2 phosphorylation in PKC- or endocytosis-dependent and EGFR-, src-, or ß-arrestin2-independent.

Asymmetric voltage waveform for enhanced exciton generation in alternative-current field-induced electroluminescence device.

We propose an alternating current (AC) field operation scheme by using an asymmetric voltage waveform to improve the electroluminescence property of AC field-induced electroluminescence (AC-FIEL) devices. Hole injection and transport can be improved by carbon nanotubes (CNT) doping into the emission layer of an AC-FIEL structure operated by a single electrode for AC-responsive alternating carrier injections. However, under an AC operation, highly unbalanced charge transports are inevitably present in CNT-doped AC-FIEL devices due to faster carrier paths through CNTs. Compared with symmetric waveform, asymmetric waveform can be adjusted to allow longer relative duty time for faster carriers in which the luminance level of CNT-doped AC-FIEL devices can be improved by 1.4 times at the same device structure and operation frequency condition.

Eukaryotic translation initiation factors as promising targets in cancer therapy.

The regulation of the translation of messenger RNA (mRNA) in eukaryotic cells is critical for gene expression, and occurs principally at the initiation phase which is mainly regulated by eukaryotic initiation factors (eIFs). eIFs are fundamental for the translation of mRNA and as such act as the primary targets of several signaling pathways to regulate gene expression. Mis-regulated mRNA expression is a common feature of tumorigenesis and the abnormal activity of eIF complexes triggered by upstream signaling pathways is detected in many tumors, leading to the selective translation of mRNA encoding proteins involved in tumorigenesis, metastasis, or resistance to anti-cancer drugs, and making eIFs a promising therapeutic target for various types of cancers. Here, we briefly outline our current understanding of the biology of eIFs, mainly focusing on the effects of several signaling pathways upon their functions and discuss their contributions to the initiation and progression of tumor growth. An overview of the progress in developing agents targeting the components of translation machinery for cancer treatment is also provided. Video abstract.

Evaluation of treatment with a combination of mycophenolate mofetil and prednisolone in dogs with meningoencephalomyelitis of unknown etiology: a retrospective study of 86 cases (2009-2017).

BACKGROUND: Combination therapy with glucocorticoids and adjunctive immunomodulating drugs has been generally accepted as a standard treatment regimen for meningoencephalomyelitis of unknown etiology (MUE). We hypothesized that treatment with MMF as an adjunctive agent along with glucocorticoids would be effective and well-tolerated protocol in dogs with MUE. Eighty-six dogs with MUE between May 2009 and June 2017 were included (59 females and 27 males; mean age of 5.93 years; mean body weight of 3.83 kg). The medical records of dogs with MUE treated with prednisolone and MMF were retrospectively evaluated to determine the therapeutic response, survival time, and treatment-related adverse effects. RESULTS: A partial or complete response (CR) was recorded for 75 dogs. The overall median survival time from the initiation of treatment was 558 days. Dogs that showed CR with no relapse over the treatment period (from diagnosis to death) had significantly longer median survival times. A significantly higher mortality hazard ratio of 4.546 was recorded in dogs that failed to achieve CR. The interval between the onset of clinical signs and the clinical presentation was not significantly associated with CR, relapse rate, and survival time. Adverse effects included gastrointestinal upsets in 26 dogs (30.23%), sporadic infections in 17 dogs (19.77%), and pancreatitis in seven dogs (8.14%). CONCLUSIONS: The results suggest that adjunctive MMF treatment for MUE is safe and comparable to other immunosuppressive protocols. The treatment should focus on the achievement of CR and preventing relapse for successful management.

Comparison of the Prognostic Factors of Fetuses With Congenital Pulmonary Airway Malformations According to Type.

OBJECTIVES: To compare the prognostic factors of fetuses with microcystic and macrocystic congenital pulmonary airway malformations (CPAMs). METHODS: We retrospectively evaluated fetuses with CPAMs at Asan Medical Center. The CPAM size, mass effect, and maximum cyst size in macrocystic CPAMs were evaluated prenatally. The adverse postnatal outcomes, including respiratory symptoms, mechanical ventilation, and surgery, were evaluated. RESULTS: In 118 cases, 2 fetal deaths and 1 neonatal death occurred. All cases of fetal hydrops and complete regression after birth were in the macrocystic and microcystic CPAM groups, respectively. Twenty-four neonates (20.7%) had respiratory symptoms, and 18 (15.5%) required mechanical ventilation. Sixty-three neonates (54.3%) underwent surgery, of whom 21 (33.3%) required surgery in the neonatal period. The maximum congenital pulmonary airway malformation volume ratio was significantly associated with all postnatal outcomes (P < .05), and the optimal cutoff values were lower for respiratory symptoms, mechanical ventilation, and neonatal surgery in the macrocystic CPAMs. The maximum cyst size was also associated with all postnatal outcomes in macrocystic CPAMs (P < .05). CONCLUSIONS: Different cutoff values for the maximum congenital pulmonary airway malformation volume ratio should be applied according to the CPAM type for the prediction of postnatal outcomes. The maximum cyst size can also be a useful prognostic factor in macrocystic CPAMs.

Intranasal MSC-derived A1-exosomes ease inflammation, and prevent abnormal neurogenesis and memory dysfunction after status epilepticus.

Status epilepticus (SE), a medical emergency that is typically terminated through antiepileptic drug treatment, leads to hippocampus dysfunction typified by neurodegeneration, inflammation, altered neurogenesis, as well as cognitive and memory deficits. Here, we examined the effects of intranasal (IN) administration of extracellular vesicles (EVs) secreted from human bone marrow-derived mesenchymal stem cells (MSCs) on SE-induced adverse changes. The EVs used in this study are referred to as A1-exosomes because of their robust antiinflammatory properties. We subjected young mice to pilocarpine-induced SE for 2 h and then administered A1-exosomes or vehicle IN twice over 24 h. The A1-exosomes reached the hippocampus within 6 h of administration, and animals receiving them exhibited diminished loss of glutamatergic and GABAergic neurons and greatly reduced inflammation in the hippocampus. Moreover, the neuroprotective and antiinflammatory effects of A1-exosomes were coupled with long-term preservation of normal hippocampal neurogenesis and cognitive and memory function, in contrast to waned and abnormal neurogenesis, persistent inflammation, and functional deficits in animals receiving vehicle. These results provide evidence that IN administration of A1-exosomes is efficient for minimizing the adverse effects of SE in the hippocampus and preventing SE-induced cognitive and memory impairments.

Comparative interactome analysis reveals distinct and overlapping properties of Raf family kinases.

The three mammalian Raf proteins (A-Raf, B-Raf, and C-Raf) are key components of the MAPK pathway. Although diverse functions have been proposed for Raf kinases, it is still not clear how interacting proteins contribute to differences in the signaling functions of the three Raf kinases. Here, we report the comparative interactomes of the three Raf kinases under serum-starved and EGF-stimulated conditions. We identified nearly 400 novel interacting proteins; some interacted with all three isoforms while others interacted exclusively with one or two. Comparing the interactomes of the three Raf kinases under different conditions revealed Raf proteins perform distinct functions through specific interactions. Our interactome data help define the differences between the three Raf kinases and may uncover new functions or regulatory mechanisms. Knowledge of Raf kinase protein-protein interactions will help us to investigate the function of specific pathways in the future.

Chromatographically isolated CD63+CD81+ extracellular vesicles from mesenchymal stromal cells rescue cognitive impairments after TBI.

Extracellular vesicles (EVs) secreted by cells present an attractive strategy for developing new therapies, but progress in the field is limited by several issues: The quality of the EVs varies with the type and physiological status of the producer cells; protocols used to isolate the EVs are difficult to scale up; and assays for efficacy are difficult to develop. In the present report, we have addressed these issues by using human mesenchymal stem/stromal cells (MSCs) that produce EVs when incubated in a protein-free medium, preselecting the preparations of MSCs with a biomarker for their potency in modulating inflammation, incubating the cells in a chemically defined protein-free medium that provided a stable environment, isolating the EVs with a scalable chromatographic procedure, and developing an in vivo assay for efficacy of the cells in suppressing neuroinflammation after traumatic brain injury (TBI) in mice. In addition, we demonstrate that i.v. infusion of the isolated EVs shortly after induction of TBI rescued pattern separation and spatial learning impairments 1 mo later.

Percutaneous full-endoscopic anterior transcorporeal cervical discectomy and channel repair: a technique note report.

BACKGROUND: Compared to anterior cervical discectomy and fusion (ACDF), cervical motion segment and disc was retained through anterior transcorporeal herniotomy (ATH). But surgical field and manipulation in traditional ATH was restricted by the narrow channel. Percutaneous full-endoscopic transdiscal cervical discectomy is a minimally invasive and functional spine surgery. However, significant loss of intervertebral disc height was inevitable. This study was done to illustrate the feasibility, safety, and efficacy and present our surgical experience of percutaneous full-endoscopic anterior transcorporeal cervical discectomy (PEATCD) and channel repair (CR) for the treatment of cervical disc herniation (CDH). METHODS: Four patients with CDH were chosen to undergo PEATCD and CR with a follow-up care for at least 22 months. The visual analogue score (VAS), Japanese Orthopedic Association (JOA), and modified Macnab criteria were recorded during the postoperative periods. CT images were obtained to observe the healing of the channel at 1 week and 3 months after the operation. RESULTS: The average operating time was 83.75 min. Drainage tubes were unnecessary. No procedure-related complications occurred. The postoperative VAS and JOA scores were improved compared to those of the preoperative assessment. The clinical efficacy was excellent in 3 patients and good in 1 patient at final follow up stage according to the modified Macnab criteria. The hernia was removed completely in all patients according to postoperative MRI. Migration of the repair implementation and collapse of the drilled vertebrae were not observed during the postoperative periods. The bony channel was nearly absent on CT images obtained at 3 months postoperative. CONCLUSION: This is the first time that the anterior transcorporeal cervical discectomy and CR have been performed simultaneously under endoscopy. Less damage to disc and the retained cervical motion segment were achieved through this method. This is a feasible, safe, and minimally invasive procedure. TRIAL REGISTRATION: Numbers: ChiCTR1800016383 . Registered 29 may 2018. Retrospectively registered. TRIAL REGISTRY: Chinese Clinical Trial Registry.

Milk Fat Globule-EGF Factor 8, Secreted by Mesenchymal Stem Cells, Protects Against Liver Fibrosis in Mice.

BACKGROUND & AIMS: Mesenchymal stem cells (MSCs) mediate tissue repair and might be used to prevent or reduce liver fibrosis. However, little is known about the anti-fibrotic factors secreted from MSCs or their mechanisms. METHODS: Umbilical cord-derived MSCs (UCMSCs) were differentiated into hepatocyte-like cells (hpUCMSCs), medium was collected, and secretome proteins were identified and quantified using nanochip-liquid chromatography/quadrupole time-of-flight mass spectrometry. Liver fibrosis was induced in mice by intraperitoneal injection of thioacetamide or CCl4; some mice were then given injections of secretomes or proteins. Liver tissues were collected and analyzed by histology or polymerase chain reaction array to analyze changes in gene expression patterns. We analyzed the effects of MSC secretomes and potential anti-fibrotic proteins on transforming growth factor ß 1 (TGFß1)-mediated activation of human hepatic stellate cell (HSC) lines (hTert-HSC and LX2) and human primary HSCs. Liver tissues were collected from 16 patients with liver cirrhosis and 16 individuals without cirrhosis (controls) in Korea and analyzed by immunohistochemistry and immunoblots. RESULTS: In mice with fibrosis, accumulation of extracellular matrix proteins was significantly reduced 3 days after injecting secretomes from UCMSCs, and to a greater extent from hpUCMSCs; numbers of activated HSCs that expressed the myogenic marker α-smooth muscle actin (α-SMA, encoded by ACTA2 [actin, alpha 2, smooth muscle]) were also reduced. Secretomes from UCMSCs, and to a greater extent from hpUCMSCs, reduced liver expression of multiple fibrotic factors, collagens, metalloproteinases, TGFß, and Smad proteins in the TGFß signaling pathways. In HSC cell lines and primary HSCs, TGFß1-stimulated upregulation of α-SMA was significantly inhibited (and SMAD2 phosphorylation reduced) by secretomes from UCMSCs, and to a greater extent from hpUCMSCs. We identified 32 proteins in secretomes of UCMSCs that were more highly concentrated in secretomes from hpUCMSCs and inhibited TGFß-mediated activation of HSCs. One of these, milk fat globule-EGF factor 8 (MFGE8), was a strong inhibitor of activation of human primary HSCs. We found MFGE8 to down-regulate expression of TGFß type I receptor by binding to αvß3 integrin on HSCs and to be secreted by MSCs from umbilical cord, teeth, and bone marrow. In mice, injection of recombinant human MFGE8 had anti-fibrotic effects comparable to those of the hpUCMSC secretome, reducing extracellular matrix deposition and HSC activation. Co-injection of an antibody against MFGE8 reduced the anti-fibrotic effects of the hpUCMSC secretome in mice. Levels of MFGE8 were reduced in cirrhotic liver tissue from patients compared with controls. CONCLUSIONS: MFGE8 is an anti-fibrotic protein in MSC secretomes that strongly inhibits TGFß signaling and reduces extracellular matrix deposition and liver fibrosis in mice.

RUVBL1, a novel C-RAF-binding protein, activates the RAF/MEK/ERK pathway to promote lung cancer tumorigenesis.

Lung cancer remains the leading cause of cancer-related deaths in the world. The RAF/MEK/ERK pathway controls many fundamental cellular functions and plays key roles in lung carcinogenesis. However, the proteins that regulate this pathway remain largely unknown. Here, we identified a novel C-RAF-binding protein, RUVBL1, which activates the RAF/MEK/ERK pathway by inhibiting phosphorylation of the C-RAF protein at serine 259. RUVBL1 expression was elevated in lung adenocarcinoma tissues. In addition, knocking out RUVBL1 effectively inhibited the proliferation and invasion of A549 cells. In vivo experiments, RUVBL1 deficiency significantly decreased the tumorigensis of lung cancer. In conclusion, we have shown that RUVBL1 could activate the RAF/MEK/ERK pathway by inhibiting phosphorylation of the C-RAF protein at serine 259, to promote lung cancer progression. Therefore, RUVBL1 could represent a novel therapeutic target for lung cancer treatment.

KAP1 inhibits the Raf-MEK-ERK pathway to promote tumorigenesis in A549 lung cancer cells.

Aberrant activation of the Raf-MEK-ERK pathway has frequently been associated with various cancers, especially lung cancer. However, the key regulators of this pathway are largely unknown. Using functional proteomics screening, we found that KAP1 interacts with c-Raf. Knocking out KAP1 decreased c-Raf phosphorylation at serine 259 and increased its phosphorylation at serine 338, which activated MEK and ERK. We detected higher KAP1 expression in lung cancer tissues than in normal peri-tumoral tissues. KAP1 knockdown arrested A549 lung cancer cells in the G0/G1 phase of the cell cycle and attenuated cell growth, metastasis, the epithelial-mesenchymal transition, angiogenesis, stemness, and colony formation. Furthermore, knocking out KAP1 remarkably increased the susceptibility of A549 cells to the anti-cancer drug 5-Fluorouracil, which correlated with increasing ERK phosphorylation. In vivo xenograft experiments suggested that KAP1 deficiency significantly decreases the tumorigenicity of A549 cells. Taken together, our findings indicate that KAP1 acts as a key module in the c-Raf-interactome complex and regulates lung cancer development through the Raf-MEK-ERK pathway. Therefore, KAP1 may represent a potential diagnosis biomarker and new treatment target for lung cancer.

Revealing A-Raf functions through its interactome.

A-Raf is a member of the Raf kinase family. Unlike B-Raf and C-Raf, the functions of A-Raf remain obscure. To gain more insight into the biological functions of A-Raf, we investigated the A-Raf interactome using proteomics. We found 132 proteins that interact with A-Raf and confirmed the interaction of 12 of these proteins with A-Raf by western blotting. Our data suggested that A-Raf regulates apoptosis, RNA catabolism, GTPase activity, and cell adhesion by interacting with proteins located in different cellular compartments. We identified all ten hallmarks of cancer in these interacting proteins, suggesting that A-Raf is involved in carcinogenesis. Our results also indicated that A-Raf may play a role in different diseases and signaling pathways. These findings have identified potential regulators of A-Raf and provide a systemic insight into its biological functions.

Emerging role of the Jun N-terminal kinase interactome in human health.

The c-Jun N-terminal kinases (JNKs) are located downstream of Ras-mitogen activated protein kinase signaling cascades. More than 20 years of study has shown that JNKs control cell fate and many cellular functions. JNKs and their interacting proteins form a complicated network with diverse biological functions and physiological effects. Members of the JNK interactome include Jun, amyloid precursor protein, and insulin receptor substrate. Recent studies have shown that the JNK interactome is involved in tumorigenesis, neuron development, and insulin resistance. In this review, we summarize the features of the JNK interactome and classify its members into three groups: upstream regulators, downstream effectors, and scaffold partners. We also highlight the unique cellular signaling mechanisms of JNKs and provide more insights into the roles of the JNK interactome in human diseases.

A-Raf: A new star of the family of raf kinases.

The Ras-Raf-MEK-MAPK (mitogen-activated protein kinase)-signaling pathway plays a key role in the regulation of many cellular functions, including cell proliferation, differentiation and transformation, by transmitting signals from membrane receptors to various cytoplasmic and nuclear targets. One of the key components of this pathway is the serine/threonine protein kinase, Raf. The Raf family kinases (A-Raf, B-Raf and C-Raf) have been intensively studied since being identified in the early 1980s as retroviral oncogenes, especially with respect to the discovery of activating mutations of B-Raf in a large number of tumors which led to intensified efforts to develop drugs targeting Raf kinases. This also resulted in a rapid increase in our knowledge of the biological functions of the B-Raf and C-Raf isoforms, which may in turn be contrasted with the little that is known about A-Raf. The biological functions of A-Raf remain mysterious, although it appears to share some of the basic properties of the other two isoforms. Recently, emerging evidence has begun to reveal the functions of A-Raf, of which some are kinase-independent. These include the inhibition of apoptosis by binding to MST2, acting as safeguard against oncogenic transformation by suppressing extracellular signal-regulated kinases (ERK) activation and playing a role in resistance to Raf inhibitors. In this review, we discuss the regulation of A-Raf protein expression, and the roles of A-Raf in apoptosis and cancer, with a special focus on its role in resistance to Raf inhibitors. We also describe the scaffold functions of A-Raf and summarize the unexpected complexity of Raf signaling.

Pirfenidone Inhibits Proliferation and Promotes Apoptosis of Hepatocellular Carcinoma Cells by Inhibiting the Wnt/ß-Catenin Signaling Pathway.

BACKGROUND Hepatocellular carcinoma (HCC) is the most important cause of cancer-related deaths worldwide. Pirfenidone is an orally available small molecule with therapeutic potential for fibrotic diseases. MATERIAL AND METHODS In this study, we analyzed the effects of different pirfenidone concentrations on the proliferation of HepG2 HCC cells using Cell Counting Kit-8 (CCK-8) and colony formation assays. Flow cytometry was performed to measure the apoptotic effects of pirfenidone on HepG2 cells. Western blot analysis was performed to detect the expression of ß-catenin and p-ß-catenin. RESULTS Pirfenidone inhibited proliferation and promoted HepG2 cell apoptosis. In addition, Western blot results indicated that pirfenidone suppressed b-catenin expression in HepG2 cells. To assess the mechanism, we treated HepG2 cells with pirfenidone, and pirfenidone plus the ß-catenin activator, SB-216763. The results revealed that SB-216763 accelerated proliferation and inhibited apoptosis in HepG2 cells treated with pirfenidone. Western blot results showed that SB-216763 upregulated ß-catenin expression in HepG2 cells treated with pirfenidone. CONCLUSIONS In conclusions, pirfenidone may be a potential drug for HCC treatment.