Apolipoprotein H-based prognostic risk correlates with liver lipid metabolism disorder in patients with HBV-related hepatocellular carcinoma.

Background: /Aim: Chronic hepatitis B patients often develop concomitant fatty liver disease, which is associated with increased risk of liver cirrhosis and hepatocarcinoma. Our previous studies have shown that apolipoprotein H (APOH) levels are gradually decreased in patients with chronic HBV infection at different stages of disease progression, and APOH deficiency disrupted hepatic lipid metabolism and caused fatty liver. We focus on the relationship between APOH and hepatocellular carcinoma (HCC) in the context of chronic HBV infection. Methods and results: APOH was downregulated at the transcriptional level in HBV-related HCC patients from open-source human liver transcriptome databases, and relatively high expression of APOH might be a favourable prognostic marker in HCC. APOH downregulation was positively associated with tumour grade and HCC subtypes. The analysis result of CHCC-HBV database showed that APOH-associated differential expression genes (DEGs) enriched in lipid metabolic pathways and downregulated APOH correlated with macrophage, neutrophil and CD8 T cell infiltration levels. Next, in vitro experiments were performed and APOH gene was silenced in HepG2.2.15 cells, an HBV producing human HCC cells. Further transcriptomic assay and analysis revealed the DEGs were enriched in cholesterol metabolism. The subsequent RT-qPCR experiments identified that CYP7A1 expression was higher upregulated in APOH silencing HepG2.2.15 cells than vehicle control cells (p < 0.05). Finally, demographic data of patients with HBV-related HCC were enrolled, and serum APOH levels were analysed using ELISA. Serum APOH levels were significantly lower in patients with HCC than in healthy controls (p < 0.05), and positively correlated with triglyceride level in healthy controls (p < 0.05). In HBV-HCC patients, serum APOH levels were positively correlated with albumin levels and negatively correlated with alkaline phosphatase (ALP), total bilirubin, and INR levels (p < 0.05). Conclusion: APOH downregulation disrupted liver lipid metabolism to potentially affect the overall survival in patients with HBV-related HCC.

Mechanism of cAMP Response Element-binding Protein 1/Death-associated Protein Kinase 1 Axis-mediated Hippocampal Neuron Apoptosis in Rat Brain Injury After Cardiopulmonary Resuscitation.

Brain injury represents a leading cause of deaths following cardiac arrest (CA) and cardiopulmonary resuscitation (CPR). This study explores the role of CREB1 (cAMP responsive element binding protein 1)/DAPK1 (death associated protein kinase 1) axis in brain injury after CPR. CA was induced by asphyxia in rats, followed by CPR. After CREB1 over-expression, the survival rate and neurological function score of rats were measured. Nissl and TUNEL staining evaluated the pathological condition of hippocampus and apoptosis of hippocampal neurons respectively. H19-7 cells were subjected to OGD/R and infected with oe-CREB1. CCK-8 assay and flow cytometry measured the cell viability and apoptosis. CREB1, DAPK1, and cleaved Caspase-3 expressions were examined using Western blot. The binding between CREB1 and DAPK1 was determined using ChIP and dual-luciferase reporter assays. CREB1 was poorly expressed while DAPK1 was highly expressed in rat hippocampus after CPR. CREB1 overexpression improved rat neurological function, repressed neuron apoptosis, and reduced cleaved Caspase-3 expression. CREB1 was enriched on the DAPK1 promoter and suppressed DAPK1 expression. DAPK1 overexpression reversed the inhibition of OGD/R-insulted apoptosis by CREB1 overexpression. To conclude, CREB1 suppresses hippocampal neuron apoptosis and mitigates brain injury after CPR by inhibiting DAPK1 expression.

Prospective, randomized, controlled, noninferiority clinical trial to evaluate the safety and efficacy of absorbable macroporous polysaccharide composites as adjunct to hemostasis during open surgery.

BACKGROUND: To address intraoperative bleeding in cardiac surgery, reducing blood transfusion requirements, is mandatory to achieve effective hemostasis. Hemostatic agents may limit localized persistent bleeding. The introduction of carboxymethyl-chitosan component into the hemostatic agent and the application of the radiation crosslinking technique maintain its capacity for achieving intraoperative hemostasis, thus increasing the clinical utility. METHODS: A prospective, noninferiority and randomized controlled clinical trial to compare the safety and efficacy of absorbable macroporous polysaccharide composites (AMPC, treatment group) with compound microporous polysaccharide hemostatic powder (CMPHP, control group) (2:1 ratio) as adjuncts to hemostasis in open surgery. The main indication was used for hemostasis in various traumatic hemorrhage areas, including cardiothoracic, vascular, and general surgery. The primary endpoint was success rate of hemostasis within 300 s (at a 10% noninferiority margin). The secondary endpoint was hemostasis time. Both endpoints were assessed in the modified intention-to-treat (MITT) population. Safety parameters were assessed. This study is fully compliant with the CONSORT statement. RESULTS: Randomized patients in AMPC and CMPHP groups were 168 and 84, respectively. In MITT population, the success rates of hemostasis within 300 s were 98.8% (163 of 165) in AMPC and 94.0% (78 of 83) in CMPHP (treatment difference 4.8% [95% CI -0.57% to 10.20%]). AMPC was thus noninferior to CMPHP. Hemostasis time (median [interquartile range]) with AMPC (87 [52.5, 180] s) was better than CMPHP (110 [54.5, 181] s). Changes in laboratory parameters over time and shifts to abnormal values were typical of surgeries and similar between two groups. No noticeable adverse effects associated with AMPC or CMPHP were observed. CONCLUSIONS: AMPC is well tolerated as topical hemostatic agent, noninferior to commercial CMPHP, and exhibits excellent safety. This study provides a novel hemostatic agent which appears to offer significant clinical advantage in various hemorrhage areas.

Comparison of clinical characteristics and mortality risk between patients with cholangiocarcinoma: A retrospective cohort study.

Background: Cholangiocarcinoma (CCA) is primary liver cancer originating from the biliary tract. The epidemiology of CCA is diverse across the globe. There are no reliably effective options for systemic therapy and CCA outcomes are poor. Herein, we examined the association between overall survival and clinical characteristics of CCA patients in our region. Methods: We included 62 CCA cases diagnosed between 2015 and 2019. Demographics, clinical history, therapeutic procedures, and concomitant diseases were abstracted. Patient survival was obtained from a household registration system. Results: The cohort was 69% male and 31% female, with 26 (42%) iCCA, 27 (44%) pCCA, and 9 (15%) dCCA. No age differences were observed between the three subtypes. Bile duct and metabolic disorders were the major concomitant diseases and showed varying associations with CCA subgroups. Serum triglycerides (TG) were higher in pCCA and dCCA than iCCA patients (p < 0.05), and TG and total cholesterol (TC) were highest among pCCA patients with cholelithiasis. Liver function appeared significant difference between iCCA, pCCA and dCCA subtypes (p < 0.01), and also in the subgroups without cholelithiasis (p < 0.01). The obstructive jaundice in pCCA patients was associated with survival time after surgery, and the presence of cholelithiasis was also another influential factor. Conclusion: We found that pCCA was more frequently associated with metabolic disorders compared to iCCA and dCCA. Postoperative survival was also associated with the degree of jaundice in pCCA compared to iCCA or dCCA. And biliary drainage is an important predictor of outcome of pCCA.

Autologous bionic tissue for inguinal hernia repair.

The prosthetic mesh, which is widely used in tension-free hernioplasty, often result in avascular stiff fibrotic scar or mesh shrinkage, causing chronic pain and infection. Here, we developed an autologous bionic tissue (ABT), which was composed of autologous bone marrow-derived mesenchymal stem cells (MSCs), poly (lactic-co-glycolic acid) (PLGA) porous scaffolds, and extracellular matrix (ECM) produced by MSCs for inguinal hernioplasty. In ABT, MSCs produced a variety of ECM composites, such as structural proteins (insoluble collagen, elastin) that provided mechanical properties, macromolecules (hyaluronic acid, glycosaminoglycan) as water and cytokines reservoir, and cell-engaging proteins (fibronectin, laminin). The above ECM composites reached the highest level in 21 days. ECM degradation related cytokines (MMP-9 and its inhibitor TIMP-1) reached the highest level on the 14th day. ECM increased the mechanical properties, elasticity, and flexibility of PLGA. Compared with the PLGA, ABT greatly inhibited inflammatory factors and promoted anti-inflammatory factors (p < 0.05), and gradually reduced the M1/M2 ratio in vivo (p < 0.05). After implantation, the thickness of tissue regeneration (p < 0.05), the number of capillaries or mature vessels (p < 0.05), the mechanical properties of ABT (p < 0.05) were greater than PLGA. MSCs and ECM could reduce the inflammation caused by PLGA, and prevent PLGA from earlier degradation and facilitate host cellular infiltration, thus ABT could greatly promote tissue regeneration in hernia repairs.

Comparative analysis of tumor-associated vascular changes following TACE alone or in combination with sorafenib treatment in HCC: A retrospective study.

The objective of the present study was to investigate the tumor-associated vascular changes in hepatocellular carcinoma (HCC) following treatment with transarterial chemoembolization (TACE) combined with sorafenib. The data of 20 patients were retrospectively analyzed. Patients underwent treatment depending on their chosen regimens (orally administered sorafenib was recommended, however the cost prevented some study articipants from selecting this course). Based on this, the patients were divided into TACE combined with sorafenib (TS) (n=10) and TACE-only treatment groups (n=10). Digital subtraction angiography images of all patients were analyzed by 2 radiologists who were blind to the type of treatment administered. The diameters of the hepatic and proper hepatic arteries, and hepatic artery branches (tumor-associated arteries), the splenic, left gastric and gastroduodenal arteries or portal veins (non-tumor-associated arteries) and the number of microvascular vessels were compared prior to and following sorafenib treatment in the TS group, between the first and second sessions of TACE in the TACE-only group and between the TS and TACE-only groups. In the TS group, the diameters of the hepatic and proper hepatic arteries, their branches and the number of microvascular vessels were significantly decreased following sorafenib treatment (P<0.05), while the diameters of the splenic, gastroduodenal and left gastric arteries were not significantly altered (P>0.05). In the TACE-only group, the diameters of the hepatic, proper hepatic, splenic, left gastric and gastroduodenal arteries were not significantly different between the first and second TACE sessions (P>0.05), while the diameters of the hepatic artery branches and the number of microvascular vessels were significantly altered (P<0.05). TACE combined with sorafenib significantly decreased the diameters of the tumor-associated arteries and the number of tumor microvascular vessels when compared with TACE treatment alone (P<0.05). No significant difference in the diameters of the portal vein and its branches between the two groups was observed (P>0.05). Treatment with TACE combined with sorafenib may significantly affect the tumor-associated vasculature compared with treatment with TACE alone in HCC.

The development of an extra-anatomic tissue-engineered artery with collateral arteries for therapeutic angiogenesis in ischemic hind limb.

To develop tissue-engineered arteries (TEAs) with collateral arteries(CAs) in ischemic hind limb goat models(IHLMs). The IHLMs created by removing femoral arteries were divided into non-treated control group(NG); non-catheter group (NCG) in which TEA was anastomosed to external iliac artery(EIA), and surrounded with collagen sponge containing autologous MSCs and VEGF-gelatin microspheres, the distal end of TEA was ligated; catheter group(CG) which received the same procedure as NCG, also received heparin infusion through catheter in EIA. TEA patency was assessed weekly by Ultrasound. The TEA and CAs were assessed by angiography, gross examination, histology and electron microscopy. In CG, TEAs remained patent for 1 month, but became partly occluded 1 week after catheter withdrawn. In NCG, TEAs were occluded 1 week after implantation. Angiography demonstrated that communication between CAs arising from the TEAs and the native vessels was established in both groups. NCG had fewer CAs than CG (P < 0.01). At 40 days, TEAs in CG demonstrated of endothelium formation, smooth muscle cells infiltration and collagen regeneration. The CG had more capillaries and mature vessels in adventia of TEAs than NCG (P < 0.01). CG group also had more vessels around TEAs than NCG (P < 0.01) or NG (P < 0.001).

Identification of mTOR as a primary resistance factor of the IAP antagonist AT406 in hepatocellular carcinoma cells.

Dysregulation of inhibitor of apoptosis (IAP) proteins (IAPs) in hepatocellular carcinoma (HCC) is often associated with poor prognosis. Here we showed that AT406, an IAP antagonist, was cytotoxic and pro-apoptotic to both established (HepG2, SMMC-7721 lines) and primary HCC cells. Activation of mTOR could be a key resistance factor of AT406 in HCC cells. mTOR inhibition (by OSI-027), kinase-dead mutation or knockdown remarkably enhanced AT406-induced lethality in HCC cells. Reversely, forced-activation of mTOR by adding SC79 or exogenous expressing a constitutively active S6K1 (T389E) attenuated AT406-induced cytotoxicity against HCC cells. We showed that AT406 induced degradation of IAPs (cIAP-1 and XIAP), but didn't affect another anti-apoptosis protein Mcl-1. Co-treatment of OSI-027 caused simultaneous Mcl-1 downregulation to overcome AT406's resistance. Significantly, shRNA knockdown of Mcl-1 remarkably facilitated AT406-induced apoptosis in HCC cells. In vivo, AT406 oral administration suppressed HepG2 tumor growth in nude mice. Its activity was potentiated with co-administration of OSI-027. We conclude that mTOR could be a key resistance factor of AT406 in HCC cells.

Therapeutic angiogenesis using basic fibroblast growth factor in combination with a collagen matrix in chronic hindlimb ischemia.

Although therapeutic angiogenesis by angiogenic cytokines is a feasible strategy to improve regional blood flow in ischemic regions, the optimal delivery mode needs to be established. Here we designed a complex of collagen matrix (CM) and basic fibroblast growth factor (bFGF) and evaluated its proangiogenic effect in ischemic hindlimbs. The bFGF-CM was prepared using lyophilization. The morphology, porosity and toxicity of CM were examined. The bFGF releasing profile and bioactivity of released bFGF were assessed. bFGF-CM was intramuscularly implanted into the rabbit ischemic hindlimb model. Oxygen saturation parameters (OSP) of ischemic hindlimbs was measured to evaluate the extremity perfusion at intervals. Histological examination was performed to evaluate the level of angiogenesis. The CM and bFGF-CM were of identical multiporous structure lacking cytotoxicity. The releasing profile lasted 10 days and the released bFGF remained bioactive. OSP in bFGF-CM group was significantly higher than that in CM, bFGF and ischemic groups at 2 and 4 weeks. The number of capillaries and mature vessels in bFGF-CM group were significantly greater than that in untreated control, CM and bFGF groups. Therefore, bFGF-CM enables the safe and effective long-term release of bFGF with improved angiogenesis in ischemic hindlimbs compared with CM devoid of bFGF.

Abdominal hernia repair with a decellularized dermal scaffold seeded with autologous bone marrow-derived mesenchymal stem cells.

Surgeons usually use synthetic polymer meshes for abdominal wall hernia repair. However, synthetic polymer meshes exhibit a lack of growth and related complications. In this study, we produced a tissue-engineered patch for abdominal hernia repair. Autologous bone-marrow-derived mesenchymal stem cells (BMSCs) were isolated and proliferated in vitro; decellularized dermal scaffolds (DSs) were prepared using enzymatic process; and then BMSCs were seeded onto the DSs for the construction of tissue-engineered patches. Under general anesthesia, rabbits underwent creation of abdominal wall defects and which were repaired with BMSC-seeded DSs, acellular DSs, and skin sutures only, respectively. Animals were sacrificed after 2 months for assessing the histological and gross examination. Abdominal hernias were absent in animals repaired with cell-seeded group, and abdominal hernias or bulges appeared in all animals repaired with acellular group. All the animals that were not repaired died within 10 days. The cell-seeded implants were thicker and indicated good angiogenesis compared with that of the acellular implants, both in histological and gross examination. The tissue-engineered patches prepared with BMSCs seeding on DSs can be used for abdominal wall hernia repair.

Preparation of decellularized and crosslinked artery patch for vascular tissue-engineering application.

There is an urgent clinical need of tissue-engineering (TE) vascular grafts, so this study was for developing a fast and simple way of producing TE vascular scaffold. The TE vascular scaffold was prepared with pepsin, DNase and RNase enzymatic decellularization and crosslinked with 0.1, 1, 5% glutaraldehyde (GA), respectively. The samples were underwent analyses of burst pressure; suture strength; cytotoxicity; enzymatic degradation in vitro; degradation in vivo; rehydration; biocompatibilities detected with hematoxylin and eosin (H&E), scan electron microscope, immunohistochemistry both in vivo and in vitro; macrophage infiltration and calcification using Von Kossa staining. After being decellularized the scaffold had a complete removal of cellular components, an intact collagen structure. The burst pressure and suture strength were similar to native artery. 0.1% GA crosslinked scaffold showed less cytotoxicity than 1 and 5% GA groups (P < 0.05) and was resistance to enzymatic degradation in vitro. Once being implanted, 0.1% GA group was resistant to degradation and formed endothelium, smooth muscle and adventitia with few macrophages infiltration. However, there appeared calcification in implants compared with that in native artery. This study demonstrated that DVPs producing methods by enzymatic decellularizing and crosslinking with 0.1% GA could be used for clinical TE vascular graft manufacture.

[Mechanism of combined use of cyclopamine and hydroxycamptothecin in inducing the apoptosis of human oral squamous cell carcinoma cell line].

OBJECTIVE: To study the mechanism underlying the effect of combined use of cyclonpamine and hydroxycamptothecin in inducing the apoptosis of human oral squamous cell carcinoma cell line (OSCC) HSQ-89. METHODS: CCK8 assay was used to investigate the inhibitory effect of cyclopamine on HSQ-89 cells. Flow cytometry (FCM) was employed to examine the cell apoptosis following combined treatment with cyclonpamine and hydroxycamptothecin. Reverse transcription polymerase chain reaction (RT-PCR) was applied to detect the mRNA expressions of Bcl-2, Bcl-xl, and Bid in HSQ-89 cells after the treatments. RESULTS: Combined treatment with cyclonpamine and hydroxycamptothecin significantly inhibited the cell proliferation compared with hydroxycamptothecin treatment alone, also resulting in a significantly higher apoptosis rate of the cells (P<0.05). The mRNA level of Bcl-2 was significantly decreased after the treatments, especially after the combined treatment. Cyclopamine produced no significant effect on the mRNA levels of Bcl-xl and Bid in the cells. CONCLUSION: The combined use of cyclopamine and hydroxycamptothecin significantly down-regulates the expression on of bcl-2 to induce the apoptosis of human OSCC cell line HSQ-89.

The development of a tissue-engineered artery using decellularized scaffold and autologous ovine mesenchymal stem cells.

Alternatives to using native arteries in vascular surgery are urgently needed. Vessels made from synthetic polymers have shortcomings such as thrombosis, rejection, intimal hyperplasia, calcification, infection, chronic inflammation and no growth potential. Tissue-engineered blood vessels (TEBV) may overcome these problems. We developed a tissue-engineered artery using autologous bone marrow derived mesenchymal stem cells (MSCs) and a decellularized arterial scaffold. Vascular smooth muscle cell (SMCs)-like cells and endothelial cell (ECs)-like cells were differentiated from MSCs in vitro. We constructed TEBV by seeding these autologous cells onto decellularized ovine carotid arteries and interposed into the carotid arteries in an ovine host models. The scaffold retained the main structural components of a blood vessel, such as collagen and elastin. The TEBVs were patent, anti-thrombogenic, and mechanically stable for 5 months in vivo, whereas non-seeded grafts occluded within 2 weeks. Histological, immunohistochemical, and electron microscopic analyses of the TEBVs demonstrated the existence of endothelium, smooth muscle and the presence of collagen and elastin both at 2 and 5 months, respectively. MSCs labeled with a fluorescent dye prior to implantation were detected in the harvested TE artery 2 months after implantation, indicating that the MSCs survived and contributed to the vascular tissue regeneration. Therefore, TEBVs can be assembled from autologous MSCs and decellularized bioscaffold.

[Epigallocatechin-3-gallate induces G1 phase cell cycle arrest in KB cells].

OBJECTIVE: To explore the effects of epigallocatechin-3-gallate (EGCG) on the proliferation of human oral epithelial cancer cell line KB cells and the molecular mechanisms. METHOD: KB cells were treated with various concentrations of EGCG for 24 or 48 h. MTT assay was used to test the cell viability. The changes of cell cycle in KB cells treated with EGCG for 48 h were analyzed using flow cytometry. The expressions of cyclin A, cyclin D1 and cyclin E were detected by RT-PCR and Western blotting. RESULT: The viability of KB cells treated with various concentrations of EGCG (25, 50, 100, 200, 400, and 800 micromol/L) for 48 h were decreased to (85.4-/+2.4)%, (80.4-/+2.8)%, (51.5-/+4.5)%, (30.2-/+1.9)%, (25.3-/+1.5)%, (20.0-/+1.1)%, respectively, showing significant difference from that of the control group [(100.0-/+2.2)%, P<0.05). EGCG decreased the viabilities of KB cells in a dose-dependent manner. Flow cytometry demonstrated that treatment with EGCG significantly increased the cell percentage in sub-G1 phase, which was (73.5-/+4.4)% after a 48-h EGCG treatment, significantly different from that in the control group [(47.3-/+3.5)%, P<0.05). EGCG-induced G1 phase arrest was correlated to the down-regulation of cyclin A and cyclin E. CONCLUSION: EGCG inhibits the proliferation of KB cells by inducing G1 phase arrest, which involves the downregulation of cyclin E.

[Epigallocatechin-3-gallate induces apoptosis in human hepatocellular carcinoma cells].

OBJECTIVE: To investigate the effects of epigallocatechin-3-gallate (EGCG) on human hepatocellular carcinoma (HCC) cells and mechanism thereof. METHOD: Human HCC cells of the lines HepG2 and SMMC-7721 were cultured and treated with of EGCG of the concentrations of 6.25, 12.5, 25, 50, 100, 200, and 400 microg/ml respectively for 24 h and 48 h. The cell viability was assessed by 3-(4,5-dimethylthiazol-2-yl)-2, 5-diphenyl tetrazolium bromide (MTT) assay. Trypan blue staining was used to count the cells. Flow cytometry was conducted to detect the cell apoptosis. The protein levels of Bcl-2, an anti-apoptosis factor, and cyclooxygenase-2 (COX-2), an up-regulator of Bcl-2. The activities of caspase-9 and caspase-3 hat promote the apoptosis of HCC cells, were measured using colorimetric method. RT-PCR was used to detect the mRNA expression of COX-2 and Bcl-2 family. RESULTS: The viabilities of the HepG2 and SMMC-7721 cells treated with EGCG of the concentrations of 50 - 400 microg/ml for 48 h reduced to 93.8% +/- 2.8%, 62.3% +/- 5.4%, 33.9% +/- 2.5%, and 17.6% +/- 3.2% respectively, all significantly lower than that of the control group [(100.0% +/- 2.8%), all P < 0.05]; and the viabilities of the SMMC-772 cells treated with EGCG of the concentrations of 50 - 400 microg/ml for 48 h reduced to 49.6% +/- 3.5%, 30.3% +/- 3.8%, 17.7% +/- 2.2%, and 13.0% +/- 2.5% respectively, all significantly lower than that of the control group [(100.0% +/- 0.8%), all P < 0.05]. After treatment with 100 microg/ml EGCG for 24 h, 48 h, 72 h, and 96 h, the live HepG2 cell numbers were (8.0 +/- 1.5), (22.0 +/- 3.1), (37.0 +/- 5.4), and (61.0 +/- 8.7) 10(4) respectively, all significantly lower than those of the control cells [(15.0 +/- 2.5), (45.0 +/- 5.3), (86.0 +/- 11.0), and (210.0 +/- 23.0) 10(4) respectively, all P < 0.05]; and the live SMMC-7721 cell numbers were (7.0 +/- 2.2), (13.0 +/- 2.5), (20.0 +/- 3.7), and (31.0 +/- 4.0) 10(4) respectively, all significantly lower than those of the control cells [(15.0 +/- 2.5), (45.0 +/- 5.3), (86.0 +/- 11.0), and (210.0 +/- 23.0) 10(4) respectively, all P < 0.05]. The apoptotic rates of HepG2 cells treated with EGCG of the concentrations of 50, 100, and 200 microg/ml for 12 h were 8.7% +/- 0.4%, 18.1% +/- 1.1%, and 22.1% +/- 1.8% respectively, all significantly higher than that of the control group (3.3% +/- 0.3%, P < 0.05); and the apoptotic rates of SMMC-7721 cells were 5.9% +/- 0.3%, 7.8% +/- 0.6%, and 12.2% +/- 0.8% respectively, all significantly higher than that of the control group (3.7% +/- 0.4%, P < 0.05). After treatment with EGCG of the concentrations of 100 and 200 microg/ml for 12 h, the caspase-9 activities of the HepG2 cells increased to (1.8 +/- 0.4) and (2.5 +/- 0.4) respectively, both significantly higher than that of the control group (1.0 +/- 0.1, both P < 0.05); and the caspase-3 activities of the HepG2 cells increased to (2.0 +/- 0.4) and (2.8 +/- 0.5) respectively, both significantly higher than that of the control group (1.0 +/- 0.2, P < 0.05) ; and the caspase-9 activities of the SMMC-7721 cells increased to (1.7 +/- 0.4) and (2.5 +/- 0.4), both significantly higher than that of the control group (1.0 +/- 0.1, both P < 0.05), and the caspase-3 activities of the SMMC-7721 cells increased to (1.9 +/- 0.4) and (2.6 +/- 0.3) respectively, both significantly higher than that of the control group [ (1.0 +/- 0.2), both P < 0.05]. When the concentration of EGCG was over 200microg/ml, it down-regulated the expression of COX-2 and Bcl-2 in both cell lines, however, EGCG resulted in no significant changes of Bcl-xl, Bax, Bad, and Bid. CONCLUSION: EGCG induces apoptosis in HCC cells through down-regulation of COX-2 and Bcl-2 and consequently activating caspase-9 and caspase-3.

Establishment of multiplexed, microsphere-based flow cytometric assay for multiple human tumor markers.

AIM: The multiplexed, microsphere-based flow cytometric assay (MFCA) for multiple human tumor markers was established for the early screening and detection of suspected cancer patients. METHODS: Covalent coupling of capture antibodies directed against their respective tumor markers to fluorescent microspheres was performed by following the protocols recommended by a commercial corporation with some modifications. The coupling efficiency and cross-reactivity were identified by the Luminex 100 system and associated software. The standard curve was constructed by using serial dilution of recombinant tumor marker standards and was validated by comparison with ELISA for quantifying the tumor markers in serum samples. RESULTS: The identifications revealed that the coupling procedures were successful without non-specific cross-reactivity and the standard curve was highly efficient. However, it was necessary to ensure the quality control of the coupling process since slight variations in the coupling procedures could profoundly affect the density of capture reagents coupled to the microspheres and consequently adversely affect the assay precision. In addition to its multi-analyte capability, the MFCA system had definite advantages, such as higher reproducibility, greater dynamic range of measurement, and considerably less preparation time and labor over the conventional "gold standard", which was the ELISA. CONCLUSION: The successful establishment of the MFCA system for the simultaneous detection of multiple tumor markers will provide the foundation for the further study of clinical applications.

Upregulation of PTEN involved in rosiglitazone-induced apoptosis in human hepatocellular carcinoma cells.

AIM: To investigate the effects of rosiglitazone, a peroxisome proliferator-activated receptor gamma (PPARgamma) agonist, on the expression of the phosphatase and tensin homologue deleted on chromosome 10 gene (PTEN) and cell growth in hepatocellular carcinoma cells, as well as the underlying mechanisms of these effects. METHODS: RT-PCR and Western blotting analyses were performed to detect transcription and the expression of PTEN in Hep3B cells treated with rosiglitazone. 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide assay was used to evaluate cell growth. Flow cytometry, DNA fragmentation analysis, caspase enzymatic assay, and Hoechst 33258 staining were used to determine cell apoptosis. Furthermore, small interfering RNA was used to suppress PTEN expression. RESULTS: Rosiglitazone increased the expression of PTEN in a dose- and time-dependent manner through the PPARgamma-dependent signal transduction pathway. PTEN upregulation was concomitant with a decreased level of Akt phosphorylation, subsequently resulting in cell growth inhibition and apoptosis in Hep3B cells. PTEN knockdown dramatically blocked these effects of rosiglitazone. Moreover, the exposure of cells to rosiglitazone activated caspases-9 and -3 during apoptotic proceeding. CONCLUSION: Thus, upregulation of PTEN is involved in the inhibition of cell growth and the induction of cell apoptosis by rosiglitazone, suggesting that rosiglitazone may be useful in liver cancer therapy via apoptosis.

Green tea polyphenol epigallocatechin-3-gallate inhibits oxidative damage and preventive effects on carbon tetrachloride-induced hepatic fibrosis.

The aim of the study was to examine the effects of epigallocatechin-3-gallate (EGCG) on hepatic fibrogenesis and on cultured hepatic stellate cells (HSCs). The rat model of carbon tetrachloride (CCl(4))-induced hepatic fibrosis was used to assess the effect of daily intraperitoneal injections of EGCG on the indexes of fibrosis. Histological and hepatic hydroxyproline examination revealed that EGCG significantly arrested progression of hepatic fibrosis. EGCG caused significant amelioration of liver injury (reduced activities of serum alanine aminotransferase and aspartate aminotransferase). The development of CCl(4)-induced hepatic fibrosis altered the redox state with a decreased hepatic glutathione and increased the formation of lipid peroxidative products, which were partially normalized by treatment with EGCG, respectively. Moreover, EGCG markedly attenuated HSC activation as well as matrix metalloproteinase (MMP)-2 activity. In cultured stellate cell, the expression of MMP-2 mRNA and protein were substantially reduced by EGCG treatment. Concanavalin A-induced activation of secreted MMP-2 was inhibited by EGCG through the influence of membrane type 1-MMP activity. These results demonstrate that administration of EGCG may be useful in the treatment and prevention of hepatic fibrosis.

Rab23 is a potential biological target for treating hepatocellular carcinoma.

AIM: To elucidate the role of Rab23 in hepatocellular carcinoma (HCC) by assessing the expression of Rab23 in HCC tissue and in HCC cell lines. METHODS: Primary tumors (n = 100) were stained with Rab23 antibodies using immunohistochemistry and in situ hybridization in tissue microarrays. Relationships between gene expression and pathology parameters were analysed. The biological significance of Rab23 in Hep-3B cells was examined by knocking down Rab23 gene expression. We designed a pair of double-stranded RNAs against human rab23 and transfected siRNA into Hep-3B cells. Rab23 expression in these cells was examined using RT-PCR and Western blots. We investigated cell growth by MTT assays and fluorescence-activated cell sorting. RESULTS: High cytoplasmic and nuclear expression of Rab23 was found in 38 of 71 (53.5%) and in 49 of 68 HCC patients (72%) respectively, which correlated with tumor size. HCC cell lines expressed Rab23. In Hep3B cells, siRNA for Rab23 decreased Rab23 mRNA by 4.5-fold and protein expression by 2-fold. Survival rates at 24 and 48 h for Hep-3B cells transfected with siRNA were lower and about 30% Hep-3B cells were apoptotic. Knocking down rab23 suppressed Hep3B cell growth, suggesting that rab23 could play an important role in Hep3B cell growth. CONCLUSION: Rab23 is overexpressed and/or activated in HCC. Rab23 may be both a HCC predictor and a target for treating HCC.

[15-deoxy-Delta(12,14)-prostaglandin J(2) induces anoikis of hepatocellular carcinoma cells: an in vitro experiment].

OBJECTIVE: To investigate the effect of 15-deoxy-Delta(12,14)-prostaglandin J(2) (15-d-PGJ(2)) on the anoikis of hepatocellular carcinoma (HC) cells and mechanisms thereof. METHODS: Fibronectin or polyhydroxyethyl methacrylate (poly-HEMA) were coated onto tissue culture plates, cell growth status and morphological changes were detected by optical microscope. DNA fragmentation analysis and Flow cytometry were used to measure cell apoptotic activity. Western blotting analysis was performed to detect the levels of focal adhesion kinase (FAK) and phosphorylated FAK(p-FAK). Furthermore, small interfering RNA (siRNA) was used to suppress FAK expression. RESULTS: The adhesion rate of the BEL-7402 cells treated with 15-d-PGJ(2) began to decrease 12 h after the treatment, time- and dose-dependently compared with the HC cell control group (all P < 0.05); when the concentration of 15-d-PGJ2 was 20 micromol/L, the adherent cells ratio at 24 h and 48 h later were (66.0 +/- 3.6)% and (35.0 +/- 5.0)% respectively. Anoikis of BEL-7402 cells was observed by flow cytometry and DNA fragmentation analysis. Western blotting showed that the p-FAK level of the BEL-7402 cells treated with 15-d-PGJ2 for 24 h decreased dose-dependently, however, the total FAK protein did not change. CONCLUSION: 15-d-PGJ(2) induces anoikis and decreases the phosphorylated FAK expression of the hepatocellular carcinoma cells.