Ursodeoxycholic acid induces apoptosis in hepatocellular carcinoma xenografts in mice.

AIM: To evaluate the efficacy of ursodeoxycholic acid (UDCA) as a chemotherapeutic agent for the treatment of hepatocellular carcinoma (HCC). METHODS: BALB/c nude mice were randomized into four groups 24 h before subcutaneous injection of hepatocarcinoma BEL7402 cells suspended in phosphate buffered saline (PBS) into the right flank. The control group (n = 10) was fed a standard diet while treatment groups (n = 10 each) were fed a standard daily diet supplemented with different concentrations of UDCA (30, 50 and 70 mg/kg per day) for 21 d. Tumor growth was measured once each week, and tumor volume (V) was calculated with the following equation: V = (L × W(2)) × 0.52, where L is the length and W is the width of the xenograft. After 21 d, mice were killed under ether anesthesia, and tumors were excised and weighed. Apoptosis was evaluated through detection of DNA fragmentation with gel electrophoresis and the terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end labeling (TUNEL) assay. Western blot analysis was performed to determine the expression of apoptosis-related proteins BAX, BCL2, APAF1, cleaved caspase-9, and cleaved caspase-3. RESULTS: UDCA suppressed tumor growth relative to controls. The mean tumor volumes were the following: control, 1090 ± 89 mm(3); 30 mg/kg per day, 612 ± 46 mm(3); 50 mg/kg per day, 563 ± 38 mm(3); and 70 mg/kg per day, 221 ± 26 mm(3). Decreased tumor volumes reached statistical significance relative to control xenografts (30 mg/kg per day, P < 0.05; 50 mg/kg per day, P < 0.05; 70 mg/kg per day, P < 0.01). Increasing concentrations of UDCA led to increased DNA fragmentation observed on gel electrophoresis and in the TUNEL assay (control, 1.6% ± 0.3%; 30 mg/kg per day, 2.9% ± 0.5%; 50 mg/kg per day, 3.15% ± 0.7%, and 70 mg/kg per day, 4.86% ± 0.9%). Western blot analysis revealed increased expression of BAX, APAF1, cleaved-caspase-9 and cleaved-caspase-3 proteins, which induce apoptosis, but decreased expression of BCL2 protein, which is an inhibitor of apoptosis, following administration of UDCA. CONCLUSION: UDCA suppresses growth of BEL7402 hepatocellular carcinoma cells in vivo, in part through apoptosis induction, and is thus a candidate for therapeutic treatment of HCC.

[Determination of phenylethanoid glycosides in Orobanche coerulescens].

Orobanche caerulescens is an important medicinal resource in Orobanchaceae. The present study aims to establish methods for determination of acteoside, crenatoside, and total phenylethanoid glycosides in O. caerulescens, and determine the content in 15 samples to evaluate the resource utilization of this medicinal plant. The content of acteoside and crenatoside were quantitatively determined by HPLC, while total phenylpropanoid glycosides was estimated by UV-VIS spectrophotometry. According to the results, the content of acteoside was the highest in O. caerulescens, followed by crenatoside. The contents of acteoside, crenatoside, and total phenylethanoid glycosides were between 1.15% - 15.60%, 0.83% - 4.47%, and 6.78% - 27.43%, respectively, which had significant differences. The acquisition time has great influence on the content of main components of O. caerulescens. The content of phenylethanoid glycosides is higher in the samples which were collected at the flowering stage. The two determination methods were proved to be simple, accurate and reliable, and can be used to evaluate the quality and resource utilization of O. caerulescens.

Interaction of Pdcd4 with eIF4E inhibits the metastatic potential of hepatocellular carcinoma.

Oxidative stress can contribute to the development of hepatocellular carcinoma (HCC) ability of the carcinoma. It has been found that oxidative stress stimulates the phosphorylation of eIF4E primarily through mitogen-activated protein kinase (MAPK) pathways resulting in increased protein translation. Utilizing specific inhibitors of MAPK pathways (SP600125 for c-Jun amino-terminal kinases [JNKs], PD098059 for extracellular signal-regulated kinases [ERKs], and SB203580 for p38 MAPK), we determined that it is primarily the inhibition of JNK that results in the suppression of the increase of p-eIF4E. We also found that PDCD4 inhibits JNK activity resulting in inhibition of the phosphorylation of c-Jun, one isoform of AP-1. We demonstrated that transfection with PDCD4 or inhibition of JNK by SP600125 alters the expression and phosphorylation of eIF4E in the presence of H(2)O(2). PDCD4 results in a stronger inhibitory effect than SP600125.

Protective effects of ursodeoxycholic acid against immune-mediated liver fibrosis in rats.

BACKGROUND/AIMS: Ursodeoxycholic acid (UDCA), a natural component of bile, has been synthesized to treat cholestatic liver diseases such as primary biliary cirrhosis. Broad biochemical changes in UDCA-treated patients suggest beneficial effects of UDCA beyond stimulating hepatobiliary secretion and possible efficacy of the medicine in treating cirrhosis of other causes. The aim was to explore the potential benefit of UDCA in controlling immune-mediated hepatic fibrosis. METHODOLOGY: We applied the rat experimental model of liver fibrosis induced by intraperitoneal injection of porcine serum. UDCA was administered orally during the course of the serum injections. RESULTS: Compared with the untreated group, the rats treated with UDCA ended with significantly higher body weight, lower liver weight, and lower spleen weight. The treated groups also demonstrated less severe liver fibrosis, with significantly lowered hepatic expression of type I and type III collagens, in terms of both mRNA and proteins. Moreover, serum levels of hyaluronic acid (HA), laminin (LN), type IV collagen (C IV), and type III procollagen (PC III) were also lower in the UDCA-treated animals. CONCLUSION: UDCA deters development of immune-mediated liver fibrosis by inhibiting the expression of collagen and other extracelluar matrix components.

Effect of ursodeoxycholic acid on TGF beta1/Smad signaling pathway in rat hepatic stellate cells.

BACKGROUND: Hepatic fibrosis is the key stage of the pathological progress from hepatic injury to cirrhosis. Ursodeoxycholic acid (UDCA) has been known as having significant clinical therapeutic effects on chronic liver diseases. Our research aimed to study the effect of UDCA on the signaling pathway of transforming growth factor beta1 (TGFbeta1)/Smad and discuss its possible molecular mechanisms of inhibiting hepatic fibrosis. METHODS: Rat hepatic stellate cells were cultured in vitro and randomly assigned to 4 groups. Group A was control group, with only DMEM culture medium applied, and groups B, C, D were experimental groups, with different doses of UDCA (1.0 mmol/L, 0.5 mmol/L and 0.25 mmol/L respectively) added into their DMEM culture medium for further culture of 24 hours and 48 hours. The protein expressions of TGFbeta1, TGF type I receptor, Smad3, Smad4 and Smad7 were measured by Western blotting, as well as the expressions of TGFbeta1, Smad3, Smad7 and cAMP response element (CREB) binding protein (CBP) mRNA by real-time PCR. SPSS 11.5 statistical package was adopted for data analyses. RESULTS: Compared with control group, the mRNA expressions of TGFbeta1 in the high and middle UDCA dose groups for 24 hours and 48 hours significantly decreased (P < 0.05), the protein expressions of TGFbeta1 in the two above groups for 48 hours and in the high dose group for 24 hours significantly decreased (P < 0.05). The protein and mRNA expressions of Smad3 in each UDCA dose group for 24 hours and 48 hours significantly decreased, with significant difference among different UDCA dose groups and between that of 24 hours and 48 hours observed (P < 0.05). The protein and mRNA expressions of Smad7 in the high and middle UDCA dose groups for 24 hours and 48 hours significantly increased. The CBP mRNA expression in each UDCA dose group for 24 hours and 48 hours significantly decreased (P < 0.05), with significant difference among different UDCA dose groups observed (P < 0.05). CONCLUSION: UDCA could curb the development of hepatic fibrosis through affecting the signaling pathway of TGFbeta1/Smad by inhibiting the expressions of TGFbeta1, Smad3 and CBP and increasing the expression of Smad7.

[Dynamics of hepatitis B virus covalently closed circular DNA after serum virology response].

OBJECTIVE: To explore the dynamics of hepatitis B virus covalently closed circular DNA (cccDNA) and optimal duration of treatment after serum virology response. METHODS: HBV cccDNA in liver biopsies and the serum HBV DNA were quantified by real time PCR, the serum makers were detected by enzyme-linked immunosorbent assay. RESULTS: (1) The cccDNA in biopsy samples continued to decrease after serum virology responded. (2) The longer the treatment after serum virology response, the lower the cccDNA level in liver tissue. (3) Anti-HBe positive patients had lower cccDNA in liver tissue than anti-HBe negative patients. (4) cccDNA in liver tissue was undetectable in 12 out of the 18 case anti-HBe(+) patients. Serum virology response lasted 35 months and anti-HBe(+) lasted 30 months. CONCLUSION: After serum virology responded, the longer the treatment, the lower the liver cccDNA. The cccDNA is undetectable in about 2/3 of the patients if the serum virological clearance lasts more than 35 months and anti-HBe lasts more than 30 months.

Synthetic chenodeoxycholic acid derivative, HS-1200, induces apoptosis of human hepatoma cells via a mitochondrial pathway.

We investigated whether HS-1200 has anti-proliferation effects on human hepatoma cells in vitro. Here, chromatin condensation, DNA ladder formation and proteolytic cleavage of poly (ADP-ribose) polymerase (PARP) were observed after treatment of HS-1200, indicating the occurrence of apoptotic cell death, which was associated with up-regulation of Bax, cleaved-caspase-3 and cleaved-caspase-9. Inhibition of caspase-9 rescued HS-1200-induced apoptosis. Furthermore, cells treated with HS-1200 showed a reduction in mitochondrial membrane potential (Deltapsi(m)) and caused cytochrome c release into the cytosol. The results indicated that synthetic chenodeoxycholic acid HS-1200 could induce cell apoptosis in BEL7402 human hepatoma cell line, via a Bax/cytochrome c/caspase-9 independent pathway. This study suggested that HS-1200 is potentially useful as an apoptosis inducer for the treatment of hepatocellular carcinoma.

Mechanism of apoptotic effects induced selectively by ursodeoxycholic acid on human hepatoma cell lines.

AIM: To investigate the effects of ursodeoxycholic acid (UDCA) on apoptosis and proliferation of hepatoma cell lines. METHODS: Human hepatoma cell lines HepG2 and BEL 7402 were cultured in medium supplemented with different concentrations of UDCA, normal human hepatic line L-02 was used as control. Cell proliferation, apoptosis and gene expression were detected using methyl thiazolyl tetrazolium (MTT) assay, flow cytometry, Western blot, DNA ladder assay, electron microscopy, and immunocytochemistry. RESULTS: Ursodeoxycholic acid inhibited the proliferation of HepG2 and BEL7402 cell lines in a dose-dependent manner. Ursodeoxycholic acid can change cell cycle distribution of HepG2 and BEL7402, the proportion of cells in G0-G1 phase increased whereas the proportion of S phase cells and G2-M phase cells decreased. Ursodeoxycholic acid arrested the cell cycle in G0-G1 phase by down-regulating the cell cycle related proteins cyclin D1, D3 and retinoblastoma protein (p(Rb)). The apoptotic rates of HepG2 and BEL7402 treated with UDCA (1.0 mmol/L) were significantly higher than those of control. In the HepG2 and BEL7402 treated with UDCA, expression of bcl-2 decreased whereas expression of Bax increased, the nuclear fragmentation and chromosomal condensed, cells shrank and lost attachment, apoptotic bodies and DNA ladders appeared. UDCA had no effect in inducing apoptosis on L-02 cell lines. CONCLUSION: UDCA can selectively inhibit proliferation and induce apoptosis of HepG2 and BEL7402 cell lines by blocking cell cycle and regulating the expression of Bax/bcl-2 genes.

The analysis of gamma-glutamyl transpeptidase gene in different type liver tissues.

AIM: To probe the value of gamma-glutamyl transpeptidase (GGT) messenger RNA in monitoring canceration of liver cells and for early diagnosis of hepatocellular carcinoma (HCC), by researching the types of GGT messenger RNA (GGTmRNA) in liver tissues and peripheral blood of different hepatopathy. METHODS: The three types of GGTmRNA (A, B, C) in liver tissues and peripheral blood from the patients with HCC, noncancerous hepatopathy, hepatic benign tumor, secondary carcinoma of liver, and healthy persons were detected by reverse-transcription polymerase chain reaction (RT-PCR). RESULTS: (1) In normal liver tissues, type A was predominantly found (100.00 %), type B was not found, type C was found occasionally (25.00 %). (2) The distribution of types of GGTmRNA in liver tissues with acute hepatitis, chronic hepatitis, cirrhosis, alcoholic hepatopathy was similar as in normal liver tissues (P>0.05), but type B was found in 3 of 18 patients with chronic hepatitis (16.67 %), and also in 3 of 11 patients with cirrhosis (27.27 %). (3) There was no significant difference of types of GGTmRNA between liver tissues with hepatic benign tumor, secondary carcinoma of liver and normal liver tissues (P>0.05). (4) Type B was predominant in cancerous tissues with HCC (87.5 %), the prevalence of type B in cancerous tissues was significantly higher than that in normal liver tissues (0/12) (P<0.05), but the prevalence of type A in cancerous tissues (46.88 %) was significantly lower than that in normal liver tissues (100.00 %) (P<0.05), and the prevalence of type C (6.25 %) in cancerous was the same as that in normal liver tissues (25.00 %) (P>0.05). In noncancerous tissues of livers with HCC, the main types were type A and type B, the prevalence of type A (85.71 %, 90.48 %) and type C (14.29 %, 9.52 %) in noncancerous tissues of liver with HCC was similar as that in normal liver tissues (A: 100.00 %; C: 25.00 %) (P>0.05), but the prevalence of type B (80.95 %, 76.19 %) in noncancerous tissues of livers with HCC was significantly higher than that in normal liver tissues (0/12) (P<0.05). (5) The prevalence of type B (37.5 %) in peripheral blood with HCC was higher than that in normal person (0/12) (P<0.05). In peripheral blood, type B was found in 4 of 11 cases of HCC with serum AFP negative. CONCLUSION: The shift of types of GGTmRNA from A to B in liver tissues may be closely related to the development of HCC, and the analysis of GGT gene may provide a useful tool for early diagnosis of HCC.

[Clinical impact of gamma-glutamyl transpeptidase messenger RNA subtypes on early diagnosis of hepatocellular carcinoma].

BACKGROUND & OBJECTIVE: It has been reported that hepatocellular carcinoma (HCC) expresses gamma-glutamyl transpeptidase (GGT) with unique carbohydrate moieties compared with normal liver GGT enzymes, this unique GGT was used as a marker for the diagnosis of HCC. However, the genomic changes in GGT relating to the development of HCC are not known. This study was designed to explore the relationship between alteration in GGTmRNA subtypes and the development of HCC, and to seek a new method for early diagnosis of HCC. METHODS: Three GGTmRNA subtypes (F, H, P) were determined bu using RT-PCR in normal liver tissues, diseased liver tissues without HCC, cancerous and noncancerous tissues from the livers with HCC, noncancerous tissues from secondary carcinoma of liver and peripheral blood of all cases. RESULTS: The main subtype of GGTmRNA was type F in normal liver tissues, liver tissues from noncancerous liver diseases and paracancerous tissues from secondary carcinoma of liver. The prevalence of subtype H was significantly higher in cancerous tissues, adjacent paracancerous and distal cancerous tissues from the livers with HCC than that in tissues from normal livers and noncancerous liver diseases (P < 0.05). The prevalence of subtype F in cancerous tissues was significantly lower than that in tissues from normal livers and noncancerous liver diseases (P < 0.05). Among 26 patients with HCC, GGTmRNA-H in peripheral blood was found in 12 cases. In 10 HCC patients with negative AFP, GGTmRNA-H in peripheral blood was found in 5 cases. CONCLUSIONS: The changes of GGTmRNA subtypes are closely related to the development of HCC, and the analysis of GGT genes might be a sensitive assay to monitor the hepatic cell canceration.