Search

Nonalcoholic fatty liver disease (NAFLD) can lead to hepatocellular carcinoma (HCC). The level of the transcription factor SOX2 correlates with HCC progression, but its role in fat accumulation remains unclear. Here, a high-fat diet, with and without fructose, significantly upregulated SOX2 in murine liver tissue. Treatment with free fatty acids (FFAs) and fructose upregulated SOX2 in murine FL83B hepatocytes. SOX2 overexpression or knockdown regulated triglyceride synthesis and lipid accumulation after FFA stimulation. CD36, a fatty acid transporter, and Yes-associated protein (YAP), a downstream molecule of the Hippo signaling pathway, were upregulated by FFA/fructose in vivo and in vitro. Transcriptional regulation of CD36 by SOX2 suggested the involvement of CD36 in SOX2-mediated hepatic steatosis. Thus, SOX2 may be a target to prevent NAFLD development.

Secreted frizzled-related protein 5 (SFRP5) plays a pivotal role in regulating the development of many tissues and organs, however, as an inhibitor of Wnt signaling, the role of SFRP5 in vitiligo remains unknown. Hence, we speculated that SFRP5 might be associated with melanogenesis in melanocytes by regulating Wnt signaling in vitiligo. In this study, we found that SFRP5 was overexpressed in the skin lesions of patients with vitiligo. Compared with that in normal epidermal melanocytes (PIG1), the expression of SFRP5 was increased in vitiligo melanocytes (PIG3V). To investigate the effect of SFRP5 on melanin synthesis, PIG1 cells were infected with recombinant SFRP5 adenovirus (AdSFRP5), and PIG3V cells were infected with recombinant siSFRP5 adenovirus (AdsiSFRP5). The results showed that SFRP5 overexpression inhibited melanin synthesis in PIG1 cells through downregulation of microphthalmia-associated transcription factor (MITF) and its target proteins via suppression of the Wnt/β-catenin signaling pathway. Accordingly, SFRP5 silencing increased melanin synthesis and activated the Wnt signaling pathway in PIG3V cells. Moreover, SFRP5 overexpression also downregulated the transcriptional activity of T cell factor/lymphoid enhancer factor (TCF/LEF) in PIG1 cells. Furthermore, this inhibitory effect of SFRP5 on melanin synthesis was reversed by treatment with the β-catenin agonist, SKL2001. The inhibitory action of SFRP5 in pigmentation was further confirmed in vivo using a nude mouse model. Hence, our results indicate that SFRP5 can inhibit melanogenesis in melanocytes. Additionally, our findings showed that SFRP5 plays a vital role in the development of vitiligo, and thus may serve as a potential therapeutic target for vitiligo.

Fragile X-related protein 1 (FXR1) is an RNA-binding protein that can regulate specific mRNA decay in cells. Our previous study showed that FXR1 expression was significantly decreased in trophoblasts from patients with unexplained recurrent spontaneous abortion (RSA); however, the role of FXR1 in trophoblast function during early placenta development has not been fully elucidated. In this study, we found that knockdown of FXR1 using siRNA effectively inhibited the migration of HTR-8 cells and extravillous trophoblast (EVT) outgrowth in an ex vivo extravillous explant culture model. Furthermore, through analysis of a panel of cytokines, we found that the GDF-15 protein was upregulated after knockdown of FXR1 in HTR-8/SVneo cells. This was further confirmed by western blotting and immunofluorescence in HTR-8/SVneo cells and an extravillous explant. Our data also showed that FXR1 expression was downregulated and GDF-15 was upregulated in chorionic villous tissues from RSA patients compared with those from healthy controls (HCs). Further, immunohistochemistry showed a strong expression of GDF-15 in chorionic villous tissue in the RSA group, which was mainly distributed in villous trophoblasts (CTBs) and syncytiotrophoblasts (STBs). Moreover, knockdown of GDF-15 enhanced the migration of HTR-8 cells, while overexpression of GDF-15 using plasmid or treatment with recombinant human GDF-15 protein inhibited trophoblast migration. Importantly, RNA-binding protein immunoprecipitation showed that FXR1 directly bound to the 3′-UTR of GDF-15 mRNA to promote GDF-15 mRNA decay. Together, our data provide new insight into the function of FXR1 in human placenta via regulation of GDF-15 expression in trophoblasts and suggest a possible pathological process involved in RSA.

To investigate whether exendin-4 (Ex-4), a glucagon-like peptide 1 receptor (GLP-1R) agonist, affects connexin 43 (Cx43) expression in osteoblasts, and determine the specific mechanism underlying Cx43 modulation by Ex-4. Osteoblast-like MC3T3-E1 cells were treated with Ex-4 with or without GLP-1R antagonist. We assessed Cx43 expression using RT-PCR, western blotting, and confocal microscopy; visualized intercellular communication using Lucifer yellow dye transfer assay; evaluated osteoblast differentiation using alkaline phosphatase and Alizarin red S (ARS) staining. Cx43 silencing or overexpression was investigated via RNA-interference or adenovirus infection. The mechanism underlying Cx43 regulation by Ex-4 was determined via treatment with either Src kinase inhibitor, KX2–391, Akt activator, sc79, or inhibitor, LY294002. Ex-4 treatment enhanced Cx43 expression and gap junctional intercellular communication in MC3T3-E1 cells. GLP-1R antagonist pretreatment abrogated the induction of Cx43 expression. Cx43 silencing significantly decreased ARS staining intensity in Ex-4-treated cells, whereas overexpression enhanced cell differentiation. Treatment with KX2–391 reduced both the Ex-4-induced increase of Cx43 expression and p-Akt protein levels. sc79 upregulated Cx43 expression, while LY294002 attenuated Cx43 upregulation by Ex-4. Induced Cx43 expression in osteoblasts via the Src-Akt signaling pathway illustrates the underlying mechanism for promoting osteoblast differentiation by Ex-4.

Elucidating the mechanism underlying osteoclast differentiation is important to improve our understanding of the pathophysiologies related to skeletal diseases and osteolytic metastasis in cancer. Sex-determining region Y-box containing gene 2 (SOX2), a stemness marker, is known to affect osteoblast differentiation and cancer metastasis. However, its role in osteoclastogenesis has not been investigated to date. Here, we report that SOX2 protein and mRNA expression was upregulated during osteoclast differentiation. The overexpression or knockdown of SOX2 in osteoclast precursor cells enhanced or suppressed, respectively, receptor activator of nuclear factor κB ligand (RANKL)-induced osteoclast differentiation and migration, and nuclear factor of activated T-cell c1 (NFATc1) and factor-associated suicide ligand (FASL) expression. In addition, epidermal growth factor receptor (EGFR) and extracellular signal-regulated kinase (ERK) activation were regulated by SOX2 expression; both EGFR and ERK inhibitors abrogated the SOX2 overexpression-induced increase in osteoclast differentiation and NFATc1 expression under RANKL stimulation. Overall, these results suggest SOX2 as a positive regulatory factor during osteoclast differentiation partly through the EGFR and ERK signaling pathways, highlighting a new potential target for restoring abnormal osteoclast activation.

Diet-induced obesity can cause metabolic or inflammatory damage on liver. Nonalcoholic fatty liver disease (NAFLD) begins with the fat accumulation in hepatocyte, but can lead to hepatocellular carcinoma (HCC). Sex-determining region Y-box 2 (SOX2) is a critical transcription factor involving regeneration and pluripotency. The expression level of Sox2 is correlated with progression of HCC, and anti-inflammatory effects of Sox2 in mesenchymal stem cells have been found. However, the expression of Sox2 by inflammatory cytokines in hepatocyte in NAFLD or the role of SOX2 in fat accumulation has been rarely reported. Here, we found that high-fat diet feeding, with or without high fructose in drinking water, significantly upregulated SOX2 in the livers of mice. In vitro, treatment with free fatty acids (FFAs) and fructose increased SOX2 expression in FL83B cells compared with the vehicle-treated group. Furthermore, overexpression or knockdown of SOX2 in FL83B cells promoted or suppressed, respectively, triglyceride synthesis and lipid accumulation after FFAs stimulation. The expression levels of several lipogenesis-related molecules were found to be altered by SOX2 expression. In addition, among several cytokines, only the treatment of tumor necrosis factor-alpha (TNFα) increased the SOX2 expression compared with the vehicle-treated control. Further, upregulation of (TNFα) by FFA/fructose was observed, and TNFα and FFA/fructose induced SOX2 expression was abolished by pretreatment of a TNFα inhibitor. Collectively, our findings suggest that TNFα-SOX2 signaling pathway in hepatocyte may be one of targets for early prevention of the development of NAFLD.

Aim The aim of this study was to assay the clinical characteristics of persistent ectopic pregnancy (PEP) and its influence on ongoing pregnancy. Methods We retrospectively reviewed 2498 patients who received salpingostomies as primary management for ectopic pregnancies from January 2004 to December 2009, using medical records and telephone inquiries. Clinical characteristics of the 52 patients (2.08%) who were diagnosed with PEP after salpingostomy were compared with those who received satisfactory treatment. The odds ratios and 95% confidential intervals were calculated for each variable by univariate and (for significantly different factors) multivariate analysis. Results Preoperatively, patients with PEP after salpingostomy significantly differed from the non-PEP patients in gestational age, mass size and pelvic adhesiolysis. Serum β-human chorionic gonadotropin levels in PEP patients were monitored after surgery, which had declined by 28.31% on postoperative day (POD) 4, 40.22% on POD 7, 51.46% on POD 10 and 53.43% on POD 21. Repeat ectopic pregnancy (REP) tended to occur more frequently in PEP patients (PEP: 5 cases, 10.20%; non-PEP: 4 cases, 2.80%; P = 0.034). Multivariate analysis showed that pelvic adhesions and PEP were the strongest independent predictors of REP. Conclusion Gestational age, mass size and pelvic adhesions were significantly correlated with PEP. PEP was an independent prognostic factor for REP. However, a multicenter study is needed to support and extend our findings.

Background: Ovarian cancer is the most common cause of gynecological cancer-associated death. Iatrogenic menopause might adversely affect the quality of life and health outcomes in young female cancer survivors. We evaluated whether postoperative hormone replacement therapy (HRT) had a negative influence on the progression-free survival (PFS) of patients with papillary serous ovarian cancer (SOC). Methods: We retrospectively reviewed the medical records of patients with papillary SOC, treated from January 1980 to December 2009, who suffered from menopause with or without HRT. Clinical characteristics of patients were compared between the two groups (HRT and non-HRT). Blood samples were collected from all the participants to detect serum cancer antigen (CA) 125. Hazard ratios with 95% confidential intervals for each variable were calculated by univariable and multivariable conditional Logistic regression analyses. Results: Among 112 identified patients, 31 were HRT users and 81 were not. The two groups did not significantly differ in median age at diagnosis ( t = 0.652, P = 0.513), International Federation of Gynecology and Obstetrics (FIGO) stage ( χ 2 = 0.565, P = 0.754), differentiation ( χ 2 = 1.728, P = 0.422), resection status ( χ 2 = 0.070, P = 0.791), relapse ( χ 2 = 0.109, P = 0.741), chemotherapy course ( t = −1.079, P = 0.282), follow-up interval ( t = 0.878, P = 0.382), or PFS ( t = 0.580, P = 0.562). Median Kupperman score at the onset of HRT was 30.81 and 12.19 after the therapy ( t = 3.302, P = 0.001). According to the analysis, the strongest independent variables in predicting PFS were FIGO stage and disease that was not optimally debulked. Conclusions: Postoperative HRT is not a prognostic factor for PFS of patients with papillary SOC. However, multicenter studies are needed to verify and extend our findings.

Bile acids entering into enterohepatic circulating are primary acids synthesized from cholesterol in hepatocyte. They are secreted actively across canalicular membrane and carried in bile to gallbladder, where they are concentrated during digestion. About 95 % BAs are actively taken up from the lumen of terminal ileum efficiently, leaving only approximately 5 % (or approximately 0.5 g/d) in colon, and a fraction of bile acids are passively reabsorbed after a series of modifications in the human large intestine including deconjugation and oxidation of hydroxy groups. Bile salts hydrolysis and hydroxy group dehydrogenation reactions are performed by a broad spectrum of intestinal anaerobic bacteria. Next, hepatocyte reabsorbs bile acids from sinusoidal blood, which are carried to liver through portal vein via a series of transporters. Bile acids (BAs) transporters are critical for maintenance of the enterohepatic BAs circulation, where BAs exert their multiple physiological functions including stimulation of bile flow, intestinal absorption of lipophilic nutrients, solubilization, and excretion of cholesterol. Tight regulation of BA transporters via nuclear receptors (NRs) is necessary to maintain proper BA homeostasis. In conclusion, disturbances of enterohepatic circulation may account for pathogenesis of gallstones diseases, including BAs transporters and their regulatory NRs and the metabolism of intestinal bacterias, etc.

Post-operative volume of subdural fluid is considered to correlate with recurrence in chronic subdural haematoma (CSDH). Information on the applications of computer-assisted volumetric analysis in patients with CSDHs is lacking.To investigate the relationship between haematoma recurrence and longitudinal changes in subdural fluid volume using CT volumetric analysis.Fifty-four patients harbouring 64 CSDHs were studied prospectively. The association between recurrence rate and CT findings were investigated.Eleven patients (20.4%) experienced post-operative recurrence. Higher pre-operative (over 120 ml) and/or pre-discharge subdural fluid volumes (over 22 ml) were significantly associated with recurrence; the probability of non-recurrence for values below these thresholds were 92.7% and 95.2%, respectively. CSDHs with larger pre-operative (over 15.1 mm) and/or residual (over 11.7 mm) widths also had significantly increased recurrence rates. Bilateral CSDHs were not found to be more likely to recur in this series. On receiver-operating characteristic curve, the areas under curve for the magnitude of changes in subdural fluid volume were greater than a single time-point measure of either width or volume of the subdural fluid cavity.Close imaging follow-up is important for CSDH patients for recurrence prediction. Using quantitative CT volumetric analysis, strong evidence was provided that changes in the residual fluid volume during the 'self-resolution' period can be used as significantly radiological predictors of recurrence.

Homeostasis of cholesterol is regulated by absorption in the intestine and synthesis in the liver. The authors previously demonstrated that OPN (osteopontin) exhibits the ability to alter hepatic cholesterol metabolism, thus affecting cholesterol gallstone formation in mice. The present study investigated the role of OPN in cholesterol gallstone formation, focusing on its effect on intestinal absorption of cholesterol. OPN gene knockout (OPN‑/‑) mice and wild‑type mice were respectively fed with a chow or lithogenic diet (LD) for 8 weeks. Following an 8‑week LD period, the incidence of gallstone, bile composition, level of serum and fecal lipids and the expression of intestinal associated genes were analyzed. OPN‑/‑ mice were protected from gallstone formation induced by 8 weeks' LD‑feeding. This protective effect from OPN deficiency was associated with alterations in bile composition, including a reduced concentration of biliary cholesterol. Additionally, plasma cholesterol level was decreased in LD‑fed OPN‑/‑ mice. The alterations primarily resulted from the decreased expression of intestinal Niemann‑Pick C1‑like (NPC1 L) 1, which is important in the intestinal absorption of cholesterol. The present study demonstrated that OPN deficiency reduced intestinal absorption of cholesterol by suppressing the expression of NPC1L1, thus protecting mice from cholesterol gallstone formation.

ObjectiveTo retrospectively analyze the rescue and treatment of pediatric patients by the Chinese Red Cross medical team during the Nepal earthquake relief.MethodsThe medical team set up a field hospital; the pediatric clinic consisted of 1 pediatrician and several nurses. Children younger than 18 years old were placed in the pediatric clinic for injury examination and treatment.ResultsDuring the 7-day period of medical assistance (the second to third week after the earthquake), a total of 108 pediatric patients were diagnosed and treated, accounting for 2.8% of the total patients. The earthquake-related injuries mainly required surgical dressing and debridement. No severe limb fractures or traumatic brain injuries were found. Infection of the respiratory tract, the gastrointestinal tract, and the skin were the most common ailments, accounting for 42.3%, 18.5%, and 16.7%, respectively, of the total treated patients.ConclusionTwo to 3 weeks after the earthquake, the admitted pediatric patients mainly displayed respiratory and gastrointestinal infections. When developing a rescue plan and arranging medical resources, we should consider the necessity of treating non–disaster-related conditions. (Disaster Med Public Health Preparedness. 2016;page 1 of 4)

Background: Osteopontin (OPN) is highly expressed in colorectal cancer (CRC) and is associated with disease progression in vivo. High levels of OPN have been demonstrated to predict low survival rates in CRC. Autophagy is a process of self-digestion, which is thought to play a significant role in carcinogenesis. However, the mechanisms of OPN's effects on CRC cell autophagy have not been elucidated. Therefore, we aimed to investigate possible mechanisms of OPN's effects on CRC autophagy. Methods: HCT116 cell proliferation, apoptosis, and migration and invasion ability were identified by cell counting k¡t-8 assay, flow cytometry, wound healing assay, and transwell chamber invasion assay, respectively. The ratios of proteins LC3-II/LC3-I, P62, and Atg7 were analyzed by Western-blot. Expressions of Beclin-1, Atg4b, Bnip3, and Vps34, both in transcriptional and translational levels, were analyzed and compared by RT-PCR and Western blot. Immunofluorescence and co-focusing experiments were used to investigate the formation of autophagosomes. Results: The results showed that OPN can promote cell proliferation, migration, and invasion, as well as inhibit cell apoptosis. It was also demonstrated that OPN could inhibit cell autophagy. Further experiments revealed that the inhibitory effect of OPN on autophagy could be reversed by blocking the p38 MAPK pathway in HCT116 cells. Conclusion: OPN is involved in HCT116 cell progression and is capable of inhibiting cell autophagy possibly by activating the p38 MAPK signaling pathway, implying that OPN could be a potential novel molecular therapeutic biomarker in patients with CRC.

Summary Background This study aimed to investigate the role of osteopontin and its receptor, integrin αv, in gallstone formation using human tissue specimens and a guinea pig lithogenic model. Material/Methods The nucleation role of osteopontin was determined in patients’ and normal gallbladder bile samples in vitro. Normal gallbladder was the control, and gallstone gallbladders were divided into group I (with normal epithelia) and group II (with degenerated epithelia) based on pathology change. Immunostaining, mRNA and protein expressions of osteopontin and integrin αv were analyzed. The animals were randomly divided into a lithogenic diet group and a normal diet group; the osteopontin mRNA expression in gallbladder and liver and osteopontin concentrations were determined. Results Osteopontin prolonged nucleation time and inhibited the pro-nucleating role induced by calcium in human bile in vitro. Immunostaining for osteopontin and integrin αv in human gallbladder tissues showed a higher reactivity in Group I than control group and Group II. The immunostaining in Group II was weaker than control group; similar results were observed for mRNA and protein expression of osteopontin and integrin αv. In the animal assay, the mRNA expression and concentration of osteopontin in gallbladder and liver gradually increased at initial stages and decreased in later stages. The concentrations of osteopontin in bile and serum of guinea pig showed similar trends. Conclusions Our results suggest that osteopontin is involved in cholesterol gallstone formation, and the role of osteopontin might correlate with integrin αv and calcium.

The topoisomerase I inhibitor, irinotecan, and its active metabolite SN-38 have been shown to induce G2/M cell cycle arrest without significant cell death in human colon carcinoma cells (HCT-116). Subsequent treatment of these G2/M-arrested cells with the cyclin-dependent kinase inhibitor, flavopiridol, induced these cells to undergo apoptosis. The goal of this study was to develop a noninvasive metabolic biomarker for early tumor response and target inhibition of irinotecan followed by flavopiridol treatment in a longitudinal study. A total of eleven mice bearing HCT-116 xenografts were separated into two cohorts where one cohort was administered saline and the other treated with a sequential course of irinotecan followed by flavopiridol. Each mouse xenograft was longitudinally monitored with proton (1H)-decoupled phosphorus (31P) magnetic resonance spectroscopy (MRS) before and after treatment. A statistically significant decrease in phosphocholine (p = 0.0004) and inorganic phosphate (p = 0.0103) levels were observed in HCT-116 xenografts following treatment, which were evidenced within twenty-four hours of treatment completion. Also, a significant growth delay was found in treated xenografts. To discern the underlying mechanism for the treatment response of the xenografts, in vitro HCT-116 cell cultures were investigated with enzymatic assays, cell cycle analysis, and apoptotic assays. Flavopiridol had a direct effect on choline kinase as measured by a 67% reduction in the phosphorylation of choline to phosphocholine. Cells treated with SN-38 alone underwent 83 ± 5% G2/M cell cycle arrest compared to untreated cells. In cells, flavopiridol alone induced 5 ± 1% apoptosis while the sequential treatment (SN-38 then flavopiridol) resulted in 39 ± 10% apoptosis. In vivo1H-decoupled 31P MRS indirectly measures choline kinase activity. The decrease in phosphocholine may be a potential indicator of early tumor response to the sequential treatment of irinotecan followed by flavopiridol in noninvasive and/or longitudinal studies. Copyright © 2011 John Wiley & Sons, Ltd.

Aim: To investigate the role of osteopontin in cholesterol gallstone formation. Methods: Nucleation time was determined in model and human gallbladder bile in vitro. Effect of osteopontin on vesicles of bile was investigated via transmission electron microscopy. The mRNA and protein expression of osteopontin were detected in human calculus and normal gallbladder tissues, and then lipid compositions of human bile were determined via commercial kits. Results: Osteopontin could prolong the nucleation time in a dose-dependent manner, and inhibit the pro-nucleation effect induced by calcium ion. Cholesterol crystal growth was inhibited by osteopontin in a dose-dependent manner in model and human gallbladder bile, but not affected by calcium. Furthermore, the formation, aggregation and fusion of vesicles were suppressed by osteopontin in model and human bile as demonstrated by transmission electron microscopy. The mRNA and protein expression of osteopontin in calculus gallbladder tissues were lower than those in normal tissues. The concentrations of cholesterol, phospholipid and bile acids, and cholesterol saturated index were higher and the contents of osteopontin and calcium, nevertheless, were found to be lower in lithogenic bile than those in normal controls. Conclusion: These findings indicated that osteopontin could inhibit the cholesterol gallstone formation as an anti-nucleation factor, which may be involved in the pathogenesis of cholesterol gallstone formation.

Proton nuclear magnetic resonance (NMR) spectra of freshly isolated human skeletal muscle samples contain creatine and phosphocreatine resonances with distinct chemical shifts that are easily visualized with magic angle spinning (MAS, spinning the sample rapidly at 54.7° with respect to the magnetic field) methods. The identification of the phosphocreatine resonance was based on two findings that: 1. the possible small dipolar coupling does not contribute to line splitting under rapid MAS, and 2. the 1H signal decreases concurrently with the phosphocreatine resonance observed in 31P NMR experiments. In the MAS 1H spectra the phosphocreatine resonance remains a singlet with a linewidth of less than 3 Hz. The creatine resonances are split into two peaks with linewidths at half height of approximately 2 and 6 Hz, respectively. The resonance with the broader linewidth represents creatine that is significantly motion-restricted and suggests that a creatine pool in muscle tissue is highly compartmentalized.

Background Nitric oxide (NO) is believed to participate in the regulation of airway clearance and non-specific cellular immunity. Recent studies have suggested that airway epithelial cells of allergic and non-allergic individuals may differ in their ability to produce this molecule. Objective The aim of this study was to detect the difference in NO production in human nasal epithelial cells between normal subjects and patients with perennial allergic rhinitis (AR), and to assess the relationship between the expression of nitric oxide synthase (NOS) isoforms and the severity of the disease. Methods Nasal epithelial cells were obtained from the inferior turbinate. The expression of mRNAs encoding constitutive endothelial NOS (eNOS) and inducible NOS (iNOS) was studied by reverse transcription-polymerase chain reaction (RT-PCR). Direct NO production in living cells was visualized and quantified by a fluorescent indicator, DAF-2 DA. Results RT-PCR analysis demonstrated that AR patients with a RAST score of 5 or 6 showed significant increases in the levels of iNOS mRNA and slight reductions in those of eNOS mRNA. Patients with a RAST score of 2–4 also revealed the same tendency however, the difference was not significant. DAF-2 DA imaging demonstrated that epithelial cells, especially the ciliated cells, produced a larger amount of NO than non-epithelial inflammatory cells. Preincubation with L-NAME resulted in an approximate 40% decrease in both groups. Conclusion These results directly indicate that nasal epithelial cells of AR patients overall produce higher levels of NO through the concomitant expression of different NOS isoforms. Continuous NO production by the epithelial cells in normal subjects further support the hypothesis that NO derived from epithelium may play dual roles in the regulation of nasal airway clearance and in the host defense. In addition, the use of DAF-2 DA provides a reliable method to visualize and quantify the direct NO production of living cells.

Objective To observe the effect of Lidan Granule (利胆冲剂, LDG) on bile lithogenic tendency and biliary 33.5 kd vesicular protein (VP) and to explore its mechanism.

Gallbladder carcinoma (GBC) is an aggressive malignancy with high mortality, mainly due to the reduced chance of curative resection and the resistance to chemotherapeutic drugs. Here, we showed that cellular Fas-associated death domain-like interleukin-1 converting enzyme inhibitory protein (c-FLIP), an anti-apoptotic protein, was over-expressed in the most of gallbladder carcinoma tissues, as judged by immunohistochemistry. Semi-quantitation was performed by determining the percentage of c-FLIP-positive cells: no positive cells (-), approximately 1% positive cells (+), approximately 30% positive cells (++), and70% positive cells (+++). Out of the 35 tissue specimens of gallbladder carcinoma, positive c-FLIP expression was found in 26 samples (6/positive+++, 13/++, 7/+), whereas negative or weak c-FLIP staining was detected in normal (1/+, 9/-) and adenomatous (2/+, 8/-) gallbladder tissues. Then, we used a small interference RNA (siRNA), which can substantially down-regulate the expression levels of c-FLIP mRNA and protein in GBC-SD and SGC-996 human gallbladder carcinoma cells, as confirmed by real-time PCR and western blot analyses. Furthermore, the combined treatment with the c-FLIP siRNA and tumor necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL) significantly induced apoptosis in gallbladder carcinoma cells, as judged by the increases in pyknosis, caspase-3/7 activities, and Annexin V-propidium iodide labeling, a marker for chromatin condensation. Thus, the siRNA-mediated down-regulation of c-FLIP profoundly enhances the sensitivity to TRAIL-induced apoptosis. In conclusion, c-FLIP expression is up-regulated in gallbladder carcinoma and the down-regulation of c-FLIP sensitizes TRAIL-induced apoptosis. The present study provides a potent strategy for the treatment of gallbladder carcinoma by targeting the c-FLIP.

Thiazolidinediones, a class of synthetic ligands to the peroxisome proliferator-activated receptor-γ, induce terminal adipocyte differentiation of 3T3 F442A cells, and have already been used as alternative therapeutic agents for the treatment of liposarcoma in clinical trials. The biochemical changes occurring in the 3T3 F442A cell line and well-differentiated liposarcoma following induction of adipocyte differentiation with the thiazolidinedione troglitazone were measured using high-resolution magic angle spinning (MAS) nuclear magnetic resonance (NMR) spectroscopy. 3T3 F442A cell differentiation was characterized by a large accumulation of intracellular triglyceride and withdrawal from the cell cycle. Phosphatidylcholine (PTC), phosphocholine (PC), myo-inositol, and glycerol were found to be possible biochemical markers for adipocyte differentiation induced by thiazolidenediones. The molar ratio of PTC to PC increased fourfold in differentiated 3T3 F442A cells compared to undifferentiated cells, suggesting a substantial increase in CTP:phosphocholine cytidylyltransferase activity with differentiation. A 2.8-fold increase in the PTC:PC ratio was observed in the lipoma-like well-differentiated liposarcoma of three patients who were treated with troglitazone when compared to liposarcoma from patients not treated with this drug. Thus, this ratio may be an NMR-detectable marker of troglitazone efficacy and response to differentiation therapy for liposarcoma. Magn Reson Med 48:602–610, 2002. © 2002 Wiley-Liss, Inc.

Introduction: Current chemotherapy for advanced soft tissue sarcoma (STS) has low overall response rates. It has been shown that the CDK4 gene is frequently amplified in some STS subtypes. Here we tested flavopiridol, a pan cyclin dependent kinase inhibitor, with STS subtypes that have amplified (dedifferentiated liposarcoma, DDLS) or non-amplified (malignant peripheral nerve sheath tumor, MPNST) CDK4. Methods: DDLS and MPNST cell lines were evaluated for cellular proliferation using CyQuant fluorescence assay, cell cycle distribution by FACS analysis and apoptosis using AnnexinV/7-AAD staining following 24h of treatment with flavopiridol. CDK4 amplification was confirmed via FISH analysis. Results: Flavopiridol inhibited proliferation of DDLS and MPNST cells with IC50s of 100 and 150nM, respectively. DDLS cells, which possess amplified CDK4, demonstrated a dose dependent increase in apoptosis with 15% and 27% cells undergoing apoptosis at 150 and 300nM flavopiridol respectively, after 24h of treatment. In contrast, the MPNST cells demonstrated a 3-fold lower apoptotic percentage compared to DDLS cells at these concentrations of flavopiridol. Lastly, the DDLS cells underwent G1 cell cycle arrest with as little as 150nM flavopiridol whereas the cell cycle distribution of the MPNST cells remained unchanged with up to 300nM flavopiridol. Conclusions: Flavopiridol has antiproliferative activity in STS cell lines. CDK4 gene amplification in dedifferentiated liposarcoma renders these cells more sensitive to flavopiridol mediated G1 cell cycle arrest and apoptosis. Our findings suggest flavopiridol to be a novel therapeutic agent for the treatment of STS, preferentially with amplified CDK4.

METHODS: Two human pancreatic cancer cell lines (AsPC-1 and S2-013) were treated with varying concentrations (0.1-100mcM) of peptide. Proliferation was measured by 3H-thymidine incorporation and cell counting at different time points (24-72h). Cell cycle analysis was determined by flow cytometry with propidium iodide DNA staining. Cell cycle-associated proteins were investigated by western blotting. Apoptosis was confirmed by annexin-binding assays.