Enhanced gemcitabine cytotoxicity with knockdown of multidrug resistance protein genes in human cholangiocarcinoma cell lines.

BACKGROUND AND AIM: Cholangiocarcinoma (CCA) is an often fatal primary cancer of the liver that tends to be resistant to chemotherapy. Multidrug resistance proteins (MRPs) contribute to the chemoresistance of these tumors. The objectives of the study were to document MRP expression profiles in two representative human intrahepatic and extrahepatic CCA cells lines (HuCCT1 and KMBC, respectively) and gemcitabine-induced cytotoxicity prior to and following MRP knockdown. METHODS: Multidrug resistance protein mRNA and protein expression were documented by real-time reverse transcription-polymerase chain reaction and western blots, respectively. MRP knockdown was achieved with lentivirus small hairpin RNA constructs. RESULTS: Prior to gemcitabine exposure, MRP1, MRP2, MRP4, MRP5, and MRP6 mRNA were expressed in HuCCT1 cells and MRP1, MRP3, MRP4, and MRP5 in KMBC cells. Following gemcitabine exposure, MRP5 and MRP6 expressions were significantly upregulated in HuCCT1 cells and MRP5 in KMBC cells. In HuCCT1 cells, although MRP5 knockdown had no effect, MRP6 knockdown significantly increased gemcitabine-induced cytotoxicity. In KMBC cells, MRP5 knockdown significantly increased gemcitabine cytotoxicity. CONCLUSIONS: Inhibition of MRP6 expression in intra-hepatic and MRP5 in extra-hepatic should be explored as potential treatments for CCA in humans.

Alterations in chemokine receptor CCR5 activity influence tumor cell biology in human cholangiocarcinoma cell lines.

INTRODUCTION AND OBJECTIVES: Intrahepatic (I-CCA) and extrahepatic (E-CCA) cholangiocarcinoma (CCA) have different growth patterns and risks for tumor metastasis. Inhibition and/or activation of the chemokine receptor CCR subclasses have been reported to alter tumor cell biology in non-CCA cancers. In this study we documented CCR expression profiles in representative human I-CCA and E-CCA cell lines and the in vitro effects of CCR antagonists and agonists on tumor cell biology. MATERIALS AND METHODS: CCR expression profiles were documented by real-time reverse transcription polymerase chain reaction; cell proliferation by WST-1; spheroid formation by sphere dimensions in anchorage-free medium; cell migration by wound healing and invasion by Transwell invasion chambers. RESULTS: All 10 CCR motifs (CCR1-10) were expressed in the I-CCA, HuCCT1 cell line and six (CCR4, 5, 6, 8, 9 and 10) in the E-CCA, KMBC cell line. In HuCCT1 cells, CCR5 expression was most abundant whereas in KMBC cells, CCR6 followed by CCR5 were most abundant. The CCR5 antagonist Maraviroc significantly inhibited cell proliferation, migration and invasion in HuCCT1 cells, and spheroid formation and invasion in KMBC cells. The CCR5 agonist RANTES had no effect on HuCCT1 cells but increased cell proliferation, migration and invasion of KMBC cells. CONCLUSION: These results suggest that CCR expression profiles differ in I-CCA and E-CCA. They also indicate that CCR5 antagonists and agonists have cell-specific effects but in general, CCR5 inactivation inhibits CCA tumor cell aggressiveness. Additional research is required to determine whether CCR5 inactivation is of value in the treatment of CCA in humans.

Hepatic Stellate Cells in Liver Fibrosis and siRNA-Based Therapy.

Hepatic fibrosis is a reversible wound-healing response to either acute or chronic liver injury caused by hepatitis B or C, alcohol, and toxic agents. Hepatic fibrosis is characterized by excessive accumulation and reduced degradation of extracellular matrix (ECM). Excessive accumulation of ECM alters the hepatic architecture leading to liver fibrosis and cirrhosis. Cirrhosis results in failure of common functions of the liver. Hepatic stellate cells (HSC) play a major role in the development of liver fibrosis as HSC are the main source of the excessive production of ECM in an injured liver. RNA interference (RNAi) is a recently discovered therapeutic tool that may provide a solution to manage multiple diseases including liver fibrosis through silencing of specific gene expression in diseased cells. However, gene silencing using small interfering RNA (siRNA) is encountering many challenges in the body after systemic administration. Efficient and stable siRNA delivery to the target cells is a key issue for the development of siRNA therapeutic. For that reason, various viral and non-viral carriers for liver-targeted siRNA delivery have been developed. This review will cover the current strategies for the treatment of liver fibrosis as well as discussing non-viral approaches such as cationic polymers and lipid-based nanoparticles for targeted delivery of siRNA to the liver.

High fiber dietary and sodium butyrate attenuate experimental autoimmune hepatitis through regulation of immune regulatory cells and intestinal barrier.

Autoimmune hepatitis (AIH) is chronic autoimmune liver disease accompanied with the imbalance of Treg/Th17 and increased intestinal permeability. We investigated the effects of a high fiber diet and sodium butyrate on the Treg/Th17 and intestinal barrier function in an experimental autoimmune hepatitis. Intraperitoneal injection of hepatic antigen (S100) was used to induce experimental autoimmune hepatitis mice model and mice were divided into normal control, S100 model control, S100 plus high fiber diet and S100 plus sodium butyrate. Serum aminotransferases and liver histology were examined. Short chain fatty acids in feces were determined by HPLC. The ratio of CD4 + C25 + Foxp3+ Treg and CD4 + IL-17 + Th17 were evaluated by flow cytometry. Tight junction proteins Zonula ocluden, Occludin and Claudin-1 were used to assess intestinal barrier function, so does Escherichia coli protein in the liver. Mice fed with either high fiber diet or sodium butyrate showed significantly lower levers of serum aminotransferases and minor liver injury compared to that of model control. Moreover, the ratio of Treg/Th17 was significantly higher in high fiber diet and sodium butyrate fed mice than that in model control. Furthermore, high fiber diet and sodium butyrate significantly increased intestinal tight junction proteins and decreased Escherichia Coli protein in the liver. In conclusion, high fiber diet and sodium butyrate can attenuate development of autoimmune hepatitis through regulation of immune regulatory cells and intestinal barrier function.

Inhibition of BMP4 and Alpha Smooth Muscle Actin Expression in LX-2 Hepatic Stellate Cells by BMP4-siRNA Lipid Based Nanoparticle.

PURPOSE: To develop and characterize vitamin A (VA)-coupled liposomes for the targeted delivery of BMP4-siRNA to hepatic stellate cells (HSC). VA was selected to increase the uptake by HSC based on their function in the storage of VA. METHODS: DOTAP/DOPE liposomes were prepared by film hydration method and their surfaces were decorated with VA. The cytotoxicity of VA-conjugated liposomes was evaluated by the WST-1 assay. Inhibition of BMP4 and α-SMA was determined by PCR and ELISA. RESULTS: VA-coated lipoplexes exhibited an average particle sizes less than 200 nm and zeta potential around +25 mV both determined using ZetaPALS. Inclusion of VA to liposomal surfaces significantly enhanced their cellular uptake without affecting cytotoxicity. VA-coupled liposomes carrying BMP4-siRNA resulted in a significant reduction in BMP4 and α-SMA at both mRNA and protein levels.  Conclusion: VA-coated liposomes were successfully designed to deliver BMP4-siRNA to specifically target HSC. The novel delivery system discussed herein may serve as a potential therapeutic strategy for the treatment of liver fibrosis in the future. This article is open to POST-PUBLICATION REVIEW. Registered readers (see "For Readers") may comment by clicking on ABSTRACT on the issue's contents page.

The Antioxidant Activity of Recombinant Rat Hepatic Fatty Acid Binding Protein T94A Variant.

PURPOSE: Liver fatty acid binding protein (FABP1) is a cytoplasmic polypeptide that transports substrates throughout the cytosol and functions as an antioxidant. A common polymorphic variant, FABP1 T94A has a minor allele frequency of 26-38%, 8.3±1.9% homozygous in the human population. The purpose of this study was to mutate and isolate recombinant rat FABP1 to the T94A variant to evaluate the mutant's antioxidant activity using in vitro studies. METHODS: Site-directed mutagenesis was used to generate a mutation in rat cDNA within a pGEX-6p-2 vector. This plasmid was transformed into competent cells and cultured for expression of FABP1 T94A mutant. The mutated protein was purified using GSTrap Fastflow columns within an ÄKTA FPLC system. A 2,7-dichlorofluorescein (DCF) assay was used to screen the T94A variant antioxidant activity. Additionally, Thiobarbituric Acid Reactive Substances (TBARS) assay was used in determining T94A mutant antioxidant activity in hydrophilic and lipophilic environments through the use of the azo compounds AAPH and MeO-AMVN, respectively and in the presence and absence of the long-chain fatty acid palmitate and α-bromo palmitate. RESULTS: Although the FABP1 T94A (20 µM) mutant significantly reduced DCF fluorescence compared to control (no protein; P< 0.001), there were no significant difference when compared to the wild-type (WT) FABP1. T94A was able to diminish the formation of malondialdehyde (MDA) in both lipophilic and hydrophilic systems. There were significant differences between T94A mutant and WT FABP1 at concentrations 1 and 10 µM (P< 0.05) in the hydrophilic milieu, however, this was not seen at 20 µM and also not seen in the lipophilic milieu at all concentrations. When T94A was pre-incubated with the long-chain fatty acids palmitate or α -bromo palmitate, MDA formation was decreased in both lipid peroxidation systems. There were no statistical differences between the WT FABP1 and T94A bound with fatty acids in both lipid peroxidation systems, however, there was a slight statistical difference when the T94A and WT FABP1 bound α-Br-PA in the AAPH lipid peroxidation system only. CONCLUSIONS: The T94A has antioxidant activity in both hydrophilic and lipophilic environments. The T94A variant of FABP1 does not have a loss of function in regard to acting as an antioxidant but the extent of function may be influenced by ligand binding. We conclude that populations having the minor T94A allele frequency would have similar ROS scavenging potential as those with nascent FABP1.

Active immunization against transforming growth factor beta1 prevents hepatic fibrosis in a rat model of liver disease.

Transforming growth factor beta1 (TGF-ß1) plays an important role in hepatic fibrogenesis. In this study, we documented the effects of active immunization against TGF-ß1 on hepatic fibrosis in an animal model of chronic liver disease. BALB/c mice were immunized against 3 different peptides of TGF-ß1 ligated into hepatitis B virus core protein (HBVc). Titers of TGF-ß1 antibodies were documented by enzyme linked immunoassays and antibody activity by cell membrane receptor binding and proliferation assays. The most immunogenic recombinant HBVc + TGF-ß1 peptide (HBVc + C) then served as a vaccine in Sprague-Dawley rats with dimethylnitrosamine-induced chronic liver disease. Hepatic fibrosis was documented by serum hyaluronic acid levels, liver histology, and reverse transcriptase polymerase chain reaction for hepatic collagen I (α1) and smooth muscle alpha actin mRNA expression. Relative to control rats vaccinated with HBVc alone, recombinant HBVc + C vaccinated animals had significantly lower serum hyaluronic acid levels, less histologic evidence of hepatic fibrosis, and reduced expression of collagen I (α1) and smooth muscle alpha actin mRNA in the liver. The results of this proof-of-concept study suggest that active immunization against TGF-ß1 is a worthwhile strategy to pursue in efforts to prevent hepatic fibrosis associated with chronic liver disease.

Long-Term, Low-Dose Exposure to Microcystin-LR Does not Cause or Increase the Severity of Liver Disease in Rodents.

BACKGROUND: Acute exposure to high concentrations of microcystin-LR (MC-LR) can cause significant hepatocyte injury. AIM: To document the effects of long-term, low-dose MC-LR exposure on hepatic inflammation and fibrosis in mice with healthy and diseased livers. MATERIAL AND METHODS: Male CD1 mice (N = 20/group) were exposed to 1.0 µg/L of MC-LR in drinking water; 1.0 µg/L MC-LR plus 300 mg/L of the hepatotoxin thioacetamide (MC-LR/TAA); or 300 mg/L TAA alone for 28 weeks. Liver biochemistry and histology were documented at the end of the study period. In addition, hepatic stellate cells (HSCs), were exposed in vitro to MC-LR (0.1-10,000 µg/L) and monitored for changes in cell metabolism, proliferation and activation. RESULTS: Liver biochemistry and histology were essentially normal in MC-LR alone exposed mice. MC-LR/TAA and TAA alone exposed mice had significant hepatic inflammation and fibrosis but the extent of the changes were similar in the two groups. In vitro, MC-LR had no effect on HSC metabolism, proliferation or activation. CONCLUSION: Long-term, low-dose exposure to MC-LR is unlikely to lead to chronic liver disease in the setting of a normal liver or exacerbate existing liver disease in the setting of ongoing hepatitis.

Increased DNase I activity in diabetes might be associated with injury of pancreas.

DNase I is an endonuclease responsible to destruction of chromatin during apoptosis. However, its role in diabetes is still unclear. With blood samples from our previous study related to type 2 diabetes, we examined the DNase I activity in the serum of these patients and the role of DNase I in the injury of pancreas was further investigated in rats and INS-1 cells. Serum and pancreatic tissues from human and rats were used for the study. Insulin resistance and diabetes were induced by high fat diet and STZ injection, respectively. DNase I activity was determined by radial enzyme-diffusion method. Expressions of DNase I and caspase-3 in pancreas were determined in rat pancreatic tissues and INS-1 cells. Apoptosis of INS-1 cells was determined by both TUNEL assay and Flow Cytometry. There was a significant elevation of DNase I activity in serum of patients with type 2 diabetes and rats with STZ injection. Moreover, increase in DNase I expression was observed in the pancreas of diabetic person and rats. Furthermore, high glucose induced both DNase I and caspase-3 expression and at the same time increased apoptosis rate of INS-1 cells. In conclusion, elevated DNase I in diabetes may be related to pancreatic injury and could be one of the causes that induce diabetes.

Hepatoprotective role of liver fatty acid binding protein in acetaminophen induced toxicity.

BACKGROUND: FABP1 has been reported to possess strong antioxidant properties. Upon successful transfection of the Chang cell line, which has undetectable FABP1 mRNA levels, with human FABP1 cDNA, the Chang cells were shown to express FABP1. Using the transfected and control (normal) Chang cells and subjecting them to oxidative stress, transfected cells were reported to be associated with enhanced cell viability. This study extends those observations by investigating the effect of FABP1 on acetaminophen (AAP)-induced hepatotoxicity. We hypothesized that presence of FABP1 would enhance cell viability compared to control cells (vector transfected cells). METHODS: Following AAP treatment of Chang FABP1 transfected and control cells, cell viability, oxidative stress, and apoptosis were evaluated using lactate dehydrogenase (LDH) release, the fluorescent probe DCF, and Bax expression, respectively. RESULTS: FABP1 cDNA transfected cells showed greater resistance against AAP toxicity than vector transfected cells. Significantly lower LDH levels (p < 0.05) were observed as were lower DCF fluorescence intensity (p < 0.05) in FABP1 cDNA transfected cells compared to vector transfected cells. FABP1 expression also attenuated the expression of Bax following AAP induced toxicity. CONCLUSION: FABP1 attenuated AAP-induced toxicity and may be considered a cytoprotective agent in this in vitro model of drug induced oxidative stress.

Randomized trial of autologous bone marrow mesenchymal stem cells transplantation for hepatitis B virus cirrhosis: regulation of Treg/Th17 cells.

BACKGROUND AND AIM: Liver cirrhosis is one of the major consequences of hepatitis B virus (HBV) infection, and transplantation of autologous bone marrow mesenchymal stem cells (ABMSCs) is one of promising therapies for patients with HBV-related liver cirrhosis (HBV-LC). However, the mechanism is unclear. The aim of the current study was to explore the role of Treg/Th17 cells in ABMSCs transplantation in patients with HBV-LC. METHODS: In this prospective study, 56 patients were enrolled and randomly assigned to transplantation group and control group. After 24-week follow-up, 39 patients completed the study (20 cases in transplantation group and 19 cases in control group). The Model for End-Stage Liver Disease scores, liver function, changes of Treg/Th17 cells, as well as related transcription factors and serum cytokines, were determined. RESULTS: Although patients in both groups showed significant improvement after Entecavir treatment, ABMSC transplantation further improved patients' liver function. Moreover, there was a significant increase in Treg cells and a marked decrease in Th17 cells in the transplantation group compared with control, leading to an increased Treg/Th17 ratio. Furthermore, mRNA levels of Treg-related transcription factor (Foxp3) and Th17-related transcription factor (RORγt) were increased and decreased, respectively. In addition, serum transforming growth factor-ß levels were significantly higher at early weeks of transplantation, while serum levels of interleukin-17, tumor necrosis factor-α, and interleukin-6 were significantly lower in patients in the transplantation group compared with control. CONCLUSION: ABMSCs transplantation was effective in improving liver function in patients with HBV-LC, which was mediated, at least in part, through the regulation of Treg/Th17 cell balance.

Regulation of transforming growth factor beta 1 gene expression by dihydropteridine reductase in kidney 293T cells.

Quinoid dihydropteridine reductase (QDPR) is an enzyme involved in the metabolic pathway of tetrahydrobiopterin (BH4). BH4 is an essential cofactor of nitric oxide synthase (NOS) and can catalyze arginine to citrulline to release nitric oxide. Point mutations of QDPR have been found in the renal cortex of spontaneous Otsuka Long Evans Tokushima Fatty (OLETF) diabetic rats. However, the role of QDPR in DN is not clear. This study investigates the effects of QDPR overexpression and knockdown on gene expression in the kidney. Rat QDPR cDNA was cloned into pcDNA3.1 vector and transfected in human kidney cells (293T). The expression of NOS, transforming growth factor beta 1 (TGF-ß1), Smad3, and NADPH oxidase were examined by RT-PCR and Western blot analyses. BH4 was assayed by using ELISA. Expression of QDPR was significantly decreased and TGF-ß1 and Smad3 were increased in the renal cortex of diabetic rats. Transfection of QDPR into 293T cells increased the abundance of QDPR in cytoplasm and significantly reduced the expression of TGF-ß1, Smad3, and the NADPH oxidases NOX1 and NOX4. Moreover, abundance of neuronal NOS (nNOS) mRNA and BH4 content were significantly increased. Furthermore, inhibition of QDPR resulted in a significant increase in TGF-ß1 expression. In conclusion, QDPR might be an important factor mediating diabetic nephropathy through its regulation of TGF-ß1/Smad3 signaling and NADPH oxidase.

Diabetes results in structural alteration of chondroitin sulfate in the urine.

PURPOSE: The assessment of the clinical significance of chondroitin sulfate in patients with type 2 diabetes mellitus (T2DM) and diabetic nephropathy (DN) for the detection of the relationship between chondroitin sulfate (CS) structure and disease. METHODS: Healthy control (n=15), type 2 diabetic patients with normalbuminuria (n=12), and patients with microalbuminuria (n=13) were enrolled in the study. Total sulfated glycosaminoglycans (GAGs) concentration in the first morning urine was evaluated by 1,9-dimethylmethylene blue method and the composition was determined by agarose gel electrophoresis. Urinary chondroitin sulfate was quantified by a combination of treatment with specific lyase digestions and separation of products by SAX-HPLC. RESULTS: GAGs concentration significantly increased in diabetic patients with microalbuminuria compared to diabetic patients with normalbuminuria. Qualitative analysis of urinary GAGs revealed the presence of chondroitin sulfate, heparan sulfate, and low-sulphated chondroitin sulphate-protein complex (LSC-PG). There was a decrease in CS and an increase in LSC-PG in the urine of patients with diabetes compared to healthy controls. Moreover, in diabetic patients, chondroitin sulfate contains more 6-sulfated disaccharide and less 4-sulfated disaccharide. There was a statistically significant difference in ratio of 6-sulfated disaccharide to 4-sulfated disaccharide among the three groups. CONCLUSIONS: GAGs were significantly increased in diabetic patients with microalbuminuria. The levels of urinary GAGs, ratio of LSC-PG/CS, as well as ratio of 6-sulfated to 4-sulfated disaccharides could be useful markers for diagnosis of patients with diabetic nephropathy.

Regulation of podocalyxin expression in the kidney of streptozotocin-induced diabetic rats with Chinese herbs (Yishen capsule).

BACKGROUND: Diabetic nephropathy is an emergent issue in China with increase in patients with type II diabetes. There are several successful Chinese herbal products for the treatment of patients with diabetic nephropathy in China. However, the mechanisms mediating the biological activity of these products are still unclear. Podocalyxin is a sialoprotein critical to maintaining integrity of filtration function of glomerulus. METHODS: By employing streptozotocin-induced diabetic rats and a Chinese herb formulation (Yishen capsule), we examined the regulation of podocalyxin expression in the kidney by Yishen capsule through immunofluorescent staining and reverse transcriptase polymerase chain reaction. RESULTS: After injection of STZ, there were significant increase in both blood glucose and urinary protein. Serum creatinine and BUN were also increased in rats with injection of STZ. Moreover, expression of podocalyxin in the glomerulus was gradually reduced after injection of STZ. There was also a loss of podocyte foot processes in the glomerular basement membrane. However, Yishen capsule or benazepril was able to restore the expression of podocalyxin and podocyte foot processes in the kidney. Although Yishen capsule could reduce urinary protein level, it has little effect on blood glucose level in the rats injected with STZ. CONCLUSIONS: Yishen capsule could attenuate the loss of podocalyxin in the glomerulus of rats injected with STZ.

The role of dorsal root ganglia activation and brain-derived neurotrophic factor in multiple sclerosis.

Multiple sclerosis (MS) is characterized by focal destruction of the white matter of the brain and spinal cord. The exact mechanisms underlying the pathophysiology of the disease are unknown. Many studies have shown that MS is predominantly an autoimmune disease with an inflammatory phase followed by a demyelinating phase. Recent studies alongside current treatment strategies, including glatiramer acetate, have revealed a potential role for brain-derived neurotrophic factor (BDNF) in MS. However, the exact role of BDNF is not fully understood. We used the experimental autoimmune encephalomyelitis (EAE) model of MS in adolescent female Lewis rats to identify the role of BDNF in disease progression. Dorsal root ganglia (DRG) and spinal cords were harvested for protein and gene expression analysis every 3 days post-disease induction (pdi) up to 15 days. We show significant increases in BDNF protein and gene expression in the DRG of EAE animals at 12 dpi, which correlates with peak neurological disability. BDNF protein expression in the spinal cord was significantly increased at 12 dpi, and maintained at 15 dpi. However, there was no significant change in mRNA levels. We show evidence for the anterograde transport of BDNF protein from the DRG to the dorsal horn of the spinal cord via the dorsal roots. Increased levels of BDNF within the DRG and spinal cord in EAE may facilitate myelin repair and neuroprotection in the CNS. The anterograde transport of DRG-derived BDNF to the spinal cord may have potential implications in facilitating central myelin repair and neuroprotection.

Prospect of bone morphogenetic protein 13 in liver diseases.

Bone morphogenetic proteins (BMPs) belong to TGF-ß superfamily and are a group of important cytokines involved in cell differentiation, proliferation and embryonic development. Multiple BMPs play important roles in several functions of vertebrates. Signaling pathway of BMPs is known to be mediated by Smad proteins, which include 8 members while Smad1, Smad5 and Smad8 are involved in BMPs signal transduction while Smad2 and Smad3 are mediated TGF-ß signal transduction. Although several BMPs such as BMP4 and BMP9 have been documented in the liver, BMP13 has not been examined in the liver. BMP13 also known as growth differentiation factor (GDF)-6 or cartilage-derived morphogenetic protein (CDMP)-2 is one of the BMPs family members. Function of BMP13 has been investigated in bone and tendon repair. It can stimulate tendon-like cell proliferation. However, our recent findings revealed that there was expression of BMP13 in the liver and its expression was modulated during metabolic disorders. The current article is to understand biological function of BMP13 especially in the liver.

Silymarin and hepatoprotection.

OBJECTIVE: To determine the hepatoprotective effect of silymarin with Chang cell cultures. Specifically, to investigate the antioxidant properties of silymarin and its protective function in reducing pro-apoptotic markers. METHODS: Intracellular free radical levels were assessed with dichlorofluorescein (DCF) fluorescence after exposing cells to an oxidative stress of 400 µmol/L H2O2 for 20 min. Levels of cellular ATP and bax expression were examined to evaluate the protective effects of silymarin. RESULTS: Silymarin significantly reduced the DCF fluorescence signal. Cell viability, assessed by the MTT assay, showed that silymarin enhanced the cell growth. Drug treatment was also associated with enhanced ATP levels, and reduced Bax and protein mRNA levels. CONCLUSION: Silymarin can function as a hepatoprotectant against free radical damage due to oxidative stress. The protective nature extends to reducing levels of pro-apoptotic Bax protein. Silymarin may be a useful adjuvant for the treatment of specific liver diseases.

Extent of extracellular signal-regulated kinases phosphorylation determines the sensitivity of hepatic stellate cells to staurosporine-induced apoptosis.

OBJECTIVE: Hepatic stellate cells (HSCs) are the principal cells responsible for the development of hepatic fibrosis and cirrhosis. During the fibrotic process, HSCs undergo proliferation and transdifferentiation from a quiescent to myofibroblast-like phenotype. The fate of myofibroblast like HSCs includes apoptosis or reversion back to a quiescent phenotype. The mechanisms involved in the apoptotic process of HSCs have yet to be determined. The purpose of the present study is to determine the effects of extracellular signal-regulated kinases (ERKs) phosphorylation on the apoptosis of HSCs induced by staurosporine. METHODS: We used Western blot and flow cytometry to detect the expression level of ERK and cell apoptosis status in four rat hepatic stellate cell lines (CFSC-8B, -2G, -3H and-5H). RESULTS: Each hepatic stellate cell line had a distinct morphology consistent with their expression level of α-SMA and that CFSC-8B cells had the highest α-SMA expression. Although all four cell types expressed similar levels of ERK1/2, phosphorylation levels were significantly higher in CFSC- 8B and CFSC-2G than in CFSC-3H and CFSC-5H cells. When CFSC-8B cells (high ERK1/2 phosphorylation) and CFSC-5H cells (low ERK1/2 phosphorylation) were employed to examine staurosporine-induced apoptosis, CFSC-8B cells were significantly more sensitive. Staurosporine further increased ERK1/2 phosphorylation in both cell lines. CONCLUSION: ERK1/2 phosphorylation in HSCs determines the sensitivity of HSCs to staurosporine-induced apoptosis.

Comparison of growth features and cancer stem cell prevalence in intrahepatic and extrahepatic cholangiocarcinoma cell lines.

Aim of the study: Intra- and extrahepatic cholangiocarcinoma (I-CCA and E-CCA respectively) exhibit different growth features that contribute to different clinical outcomes. Cancer stem cells (CSCs) influence tumor growth and thereby may be responsible for these differences. The aim of this study was to document and compare the growth features of human I-CCA and E-CCA cell lines and determine whether any differences observed could be explained by differences in the prevalence and/or stem cell surface marker (SCSM) expression profiles of CSCs within the tumor cell lines. Material and methods: Six CCA cells lines, three I-CCA and three E-CCA, were studied. Tumor cell growth features including cell proliferation, colony/spheroid formation, migration and invasion were documented. CSC prevalence and SCSM expression profiles were examined by flow cytometry. Results: I-CCA cells had significantly increased proliferative activity, shorter doubling times and were more invasive than E-CCA cells, while colony/spheroid formation and migration were similar in the two cell populations. There were no significant differences in CSC prevalence rates or SCSM expression profiles. Conclusions: These findings suggest that I-CCA cells proliferate at a more rapid rate and are more invasive than E-CCA cells but the differences cannot be explained by differences in the prevalence or SCSM expression profiles of CSCs within the tumor cell population.

Attenuation of Diabetic Nephropathy in Otsuka Long-Evans Tokushima Fatty (OLETF) Rats with a Combination of Chinese Herbs (Tangshen Formula).

Diabetic nephropathy is one of the most significant microvascular complications in patients with type 2 diabetics. The concise mechanism of diabetic nephropathy is unknown and there is no successful treatment. The objective of study was to investigate effects of Chinese herbs (Tangshen Formula) on diabetic nephropathy in Otsuka Long-Evans Tokushima Fatty (OLETF) rats. OLETF rats and LETO rats were divided into four groups: LETO control, OLETF diabetics, OLETF diabetics treated with Tangshen Formula, and OLETF diabetics treated with Monopril. Body weight, blood glucose, and 24 h urinary proteins were measured once every four weeks. Blood samples and kidney tissues were obtained for analyses of total cholesterol, triglyceride, whole blood viscosity, plasma viscosity, and pathohistological examination at 36 and 56 weeksrespectively. Untreated OLETF rats displayed diabetic nephropathy over the study period. Treatment of OLETF rats with Tangshen Formula attenuated the increases in blood glucose, body weight, 24 h urinary protein content, serum total cholesterol, whole blood viscosity and plasma viscosity at certain time. Treatment with Tangshen Formula also reduced glomerulosclerotic index and interstitial fibrotic index seen in OLETF rats. In conclusion, Tangshen Formula could attenuate the development of diabetic nephropathy in OLETF rat diabetic model.