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Cancer stem cells (CSCs) are considered to play essential roles in the process of origination, proliferation, migration, and invasion of cancer, and their properties are regulated by Wnt/β-catenin pathway. Phenethyl isothiocyanate (PEITC) is a natural product obtained from cruciferous vegetables with anticancer activities. The present study aimed to investigate the inhibitory effect and the underlying mechanisms of PEITC on colorectal CSCs. In this study, we found that PEITC can significantly reduce the size and number of colorectal cancer cell spheroids in serum-free medium. With increasing PEITC concentrations (10-40 μM), the number of spheroids was reduced to about 10% of the control group, and the percentage of CD133+ cells was decreased by about 3-16 folds. PEITC also decreased the expression of CSC markers. Meanwhile, inhibition of proliferation as well as induction of apoptosis of colorectal CSCs was observed after PEITC treatment. Furthermore, through activating Wnt/β-catenin pathway with LiCl, the inhibitory effects of PEITC on colorectal CSCs were diminished. Our data suggested that PEITC can be an effective inhibitor of colorectal CSCs by targeting Wnt/β-catenin pathway. [ABSTRACT FROM AUTHOR]

The beneficial effects of tea consumption on cancer prevention have been generally reported, while (-)-Epigallocatechin-3-gallate (EGCG) is the major active component from green tea. Cancer stem cells (CSCs) play a crucial role in the process of cancer development. Targeting CSCs may be an effective way for cancer intervention. However, the effects of EGCG on colorectal CSCs and the underlying mechanisms remain unclear. Spheroid formation assay was used to enrich colorectal CSCs from colorectal cancer cell lines. Immunoblotting analysis and quantitative real-time polymerase chain reaction were used to measure the alterations of critical molecules expression. Immunofluorescence staining analysis was also used to determine the expression of CD133. We revealed that EGCG inhibited the spheroid formation capability of colorectal cancer cells as well as the expression of colorectal CSC markers, along with suppression of cell proliferation and induction of apoptosis. Moreover, we illustrated that EGCG downregulated the activation of Wnt/β-catenin pathway, while upregulation of Wnt/β-catenin diminished the inhibitory effects of EGCG on colorectal CSCs. Taken together, this study suggested that EGCG could be an effective natural compound targeting colorectal CSCs through suppression of Wnt/β-catenin pathway, and thus may be a promising agent for colorectal cancer intervention., Competing Interests: The authors declare no conflict of interest.

Objective: To investigate the effects and related mechanisms of hepatitis B virus X (HBx) protein on cell cycle and growth in hepatocellular carcinoma., Methods: A human hepatocyte HepG2 cell line stably expressing a green fluorescent protein (GFP)-tagged HBx (HepG2/GFP-HBx cells) was used for the experiment, and HepG2 parental and HepG2/GFP cells was used as the controls. Effect of HBx on cell growth was evaluated by the MTT cell proliferation assay and on cell cycle progression by flow cytometry analysis of cells with or without treatment with 5-aza-2'-deoxycytidine (5-Aza-CdR; 5 pmol/L). Effect of HBx expression on promoter methylation status of the p16INK4A tumor-suppressor gene was detected by methylation-specific polymerase chain reaction and on p16 protein level was analyzed with western blotting., Results: The HepG2/GFP-HBx cells showed significantly higher cell proliferation at 72 hrs of culture (3.225+/-0.038 A490) than either control (HepG2: 2.012+/-0.022 A490, t = -46.86, P less than 0.001; HepG2/GFP: 2.038+/-0.029 A490, t = 42.51, P less than 0.001). The HepG2/GFP-HBx cells also showed significantly lower proportion of cells in the G0/G1 phase (16.45%+/-0.45%) than either control (HepG2: 44.81%+/-1.36%, t = -34.202, P less than 0.001; HepG2/GFP: 42.76%+/-1.58%, t = -28.88, P less than 0.001). However, 5-Aza-CdR treatment did lead to a significant amount of HepG2/GFP-HBx cells being arrested in the G0/G1 phase (33.25%+/-0.79%, t = 31.85, P less than 0.001). The p16INK4A promoter was methylated in the HepG2/GFP-HBx cells, and became demethylation after treatment with 5-Aza-CdR. However, no methylation of p16INK4A promoter was observed in both HepG2 and HepG2/GFP cells. The p16 protein level was significantly lower in the HepG2/GFP-HBx (vs. HepG2 and HepG2/GFP cells) and this level increased after treatment with 5-Aza-CdR., Conclusion: HBx protein promotes hepatocellular carcinoma cell cycle progression and growth by shortening the G0/G1 phase, and the underlying mechanism may involve inducing p16INK4A promoter methylation and downregulating p16 protein expression.

The ability of lymphocytes and macrophage-derived cytokines and chemokines to modulate the activation of stromal cells during immune responses is well-documented, but few studies have investigated whether liver myofibroblasts shape the phenotype and function of monocytes in liver disease. In the present study, Kupffer cells were demonstrated to be activated in the inflamed livers of patients with cirrhosis and be in close contact with liver myofibroblasts. The Kupffer cells from cirrhotic livers expressed significantly elevated levels of PD-L1 (also termed B7-H1), TLR4, CD80, CD32 and CD64 relative to those from normal livers. Consistent with this finding, the expression of these surface molecules was significantly upregulated in monocytes following exposure to liver myofibroblasts originating from inflamed livers. Accordingly, the liver myofibroblast-exposed monocytes exhibited a significant increase in dextran endocytosis. These data reveal that bidirectional interactions between liver myofibroblasts and Kupffer cells may function as an 'amplification loop' to enhance inflammation further in the liver. Liver myofibroblasts are central in the pathogenesis of liver diseases and should be considered as targets for the rational design of effective immune-based anti-inflammation therapies. Furthermore, it was also demonstrated that skin fibroblasts were as effective as liver myofibroblasts at inducing monocyte activation, suggesting that fibroblasts, which are numerous in the body, may represent an underrated cell population that is actively involved in immunomodulatory functions.

Objective: To investigate the relationship and clinical significances of HBeAg status with serum HBV DNA loads, model for end-stage liver disease (MELD) scores in patients with acute-on-chronic hepatitis B liver failure during terminal phase., Methods: 120 fatal patients were enrolled. At three phases of 0 -14 d, 15-28 d and 29-90 d before death, they were detected serum HBeAg, HBV DNA loads order meanwhile MELD scores were calculated., Results: In 51 patients with HBeAg positive, HBV DNA levels were (5.25 +/- 1.99), (5.45 +/- 1.47) and (6.06 +/- 1.77) log10 copies/ml while MELD scores were (30.33 +/- 5.25), (26.36 +/- 6.43) and (20.13 +/- 6.47) respectively. In 69 patients with HBeAg negative,HBV DNA loads were (5.14 +/- 1.84), (5.49 +/- 1.75 ) and (4.62 +/- 1.65) log10 copies/ml while MELD scores were 32.38 +/- 9.95, 28.17 +/- 6.82 and 26.19 +/- 5.56 in sequence. Compared with the same phase between HBeAg-positive group and HBeAg-negative group, significant differences in both HBV DNA loads and MELD scores were found only at the phase of 29-90 d (P < 0.05). In multiple comparisons among three phases, regardless of the HBeAg status,there wasn't significant difference for HBV DNA loads (P > 0.05). But increasing MELD scores are associated with the disease exacerbation and significant differences were found (P < 0.05)., Conclusions: To initiate acute-on-chronic hepatitis B liver failure, serum HBV DNA loads of HBeAg-positive patients are higher than that of HBeAg-negative ones. Once ACLF has been initiated,sustained high HBV DNA loads may promote the disease worsened and be fatal regardless of the HBeAg status.

Objective: To explore the opportunity and effect of internal general treatment added entecavir on acute-on-chronic liver failure (ACLF) of HBeAg-negative chronic hepatitis B patients in different ranges of MELD score., Methods: A total of 101 ACLF of HBeAg-negative chronic hepatitis B patients treated with internal general treatment added entecavir were divided into three groups according to the MELD score. The mortalities and HBV DNA loads during the initiation of therapy, recovery phase and in deathbed phase were studied., Results: 20 of patients with high MELD score (> or = 30) received (14.6 +/- 14.1) days treatment. The difference in MELD score between pre-(36.03 +/- 5.01) and post-treatment (39.86 +/- 5.95) was significant (t = - 2.994, P = 0.007). There was no significant difference in HBV DNA load between pre-[(4.454 +/- 1.714) copies log10/ml] and post-treatment [(3.979 +/- 1.947) copies log10/ml] (t = 2.212, P = 0.051), the mortality was 100% (20/20). 47 of patients with moderate MELD score (22-30) received (51.5 +/- 41.6) days treatment. There was no significant difference in MELD score between pre-(25.71 +/- 2.47) and post-treatment (26.18 +/- 13.32) (t = - 0.263, P = 0.794). The difference in MELD score between pre-[(6.084 +/- 1.795) copies log10/ml] and post-treatment [(3.378 +/- 2.156) copies log10/ml] was significant (t =7.148, P = 0.000), the mortality was 53.19% (25/47). 34 of patients with low MELD score (< or = 22) received (67.2 +/- 40.9) days treatment. The difference in MELD score was significant between pre-(< or = 18.85 +/- 2.72) and post-treatment (11.68 +/- 7.23) (t = 5.983, P = 0.000). There was significant difference in HBV DNA load between pre-[(5. 945 +/- 1.635) copies log10/ml] and post-treatment [(2.725 +/- 1.194) copies log10/ml] (t = 9.962, P = 0.000), the mortality was 2.94% (1/34)., Conclusions: The ACLF of HBeAg-negative chronic hepatitis B patients with a low score of MELD score (< or = 22) mostly survive with internal general treatment added entecavir. The mortality of the patients with a MELD score (22-30) is 53.19% (25/47). The patients with high MELD score (> or = 30) which almost lack the opportunity of treatment, is associated with fatal liver failure and need for emergency liver transplantation.

Objective: To observe the therapeutic effect and safety of entecavir and adefovir in the treatment of lamivudine-resistant HBeAg-negative chronic hepatitis B., Methods: Sixty-five patients with lamivudine-resistant HBeAg-negative chronic hepatitis B were randomly divided into two groups. The entecavir treatment group included 33 patients, who were administrated entecavir 1.0 mg/d. The adefovir treatment group included 32 patients, who were administrated adefovir dipivoxil 10 mg/d. Changes in serum HBV DNA, liver functions, phosphocreatine kinase, creatinine and adverse reaction were dynamically monitored., Results: At the end of the 12th, 24th, 48th week of treatment, the rates of serum ALT normalization of the entecavir treatment group were higher than that of the adefovir treatment group, but there wasn't statistically difference between two groups until the end of the 48 th week of treatment (P > 0.05). The rate of sera to turn negative for HBV DNA of the entecavir treatment group was significantly higher than that of the adefovir treatment group at the end of the 12th week. Moreover, the difference was statistically significant (P<0.05)., Conclusion: Both entecavir and adefovir dipivoxil might have a good response to lamivudine-resistant HBeAg-negative chronical hepatitis B. Entecavir could achieve better therapeutic effects.

Objective: To establish an objective, simple and sensitive scoring system to evaluate the severity of acute-on-chronic liver failure in hepatitis B., Methods: The clinical data of patients (194 survivals and 215 deaths) with acute-on-chronic liver failure in hepatitis B were collected and analyzed prospectively. 7 clinical indexes, including the hepatic encephalopathy, creatinine, prothrombin activity, serum total bilirubin, infection, the dimension of liver, the maximum depth of ascites, were scored objectively and simply from 0 to 4 points according to their severity. Then we calculated every patient's total score and divided the 409 patients into two groups: the one was 309 patients and the other is 100 patients. The first group was to establish the severity scoring system and define the cut-off-point, the second group was to test the severity scoring system., Results: The total score of the 144 patients in the survival group was 6.9 +/- 3.2, 165 patients in the dead group was 15.8 +/- 4.0, respectively. There were significant differences (P < 0.01) between the two groups. The area under ROC curve was 0.953. The cut-off-point is 9.5. The sensitivity was 0.97, the specificity was 0.82. The second group patients' total score were divided into two groups: the one is > or = 10 score and the other is < or = 9 score. The prognosis of the first group was much worse than the second group, it's mortality rate was 87.5%; the second was 2.3%. There were significant differences between the two groups (P < 0.01)., Conclusions: This scoring system was simple, sensitive and objective to evaluate the severity of acute-on-chronic liver failure in hepatitis B.

Objective: To investigate the status of vaccination against hepatitis B among postgraduate students of medical institutions of higher education in Guangzhou., Methods: HBsAg and anti-HBs in the serum samples from 1139 postgraduate students were detected by ELISA. Data on hepatitis B vaccine inoculation were investigated by using a questionnaire. Statistical analyses were performed by using SAS software., Results: The HBsAg positive rate among the 1139 postgraduate students was 2.90 percent. The HBsAg positive rates in hepatitis B vaccine inoculated (1.15 percent) and non- inoculated (21.69 percent) postgraduate students were significantly different (x2=119.11, P<0.0001). The positive rates of HBsAb between the two groups were also significantly different (x2=62.05, P<0.0001). Among the hepatitis B vaccine inoculated students, 17.31 percent were negative for HBsAb. The positive rate of HBsAb among those inoculated the vaccine within the past 3 years was higher than that among those inoculated the vaccine earlier (0-3 years vs. 4-6 year, P=0.0089) (0-3 years vs. 7-9 years, P=0.0172) (0-3 years vs. >9 years, P=0.0474). The positive rate of HBsAb among the students who received hepatitis B vaccine booster dose was higher than that of the students who did not receive any booster dose (P=0.0093)., Conclusion: With the increase of ages, the effect of vaccination for hepatitis B decreased. Male populations may be more susceptible to hepatitis B virus than female. It is necessary to monitor HBsAb levels for those who were inoculated with HBV vaccine more than 3 years ago to give booster dose in time to prevent HBV infection.

Objectives: To investigate the cases of chronic hepatitis B relapse after lamivudine withdrawal, and to find clinical characteristics and related factors to them., Methods: 46 cases of chronic hepatitis B relapse after lamivudine withdrawal were investigated and followed up. The diagnosis and the course of the diseases before therapy with lamivudine, the dosage, effects, period of treatment, the reasons for lamivudine withdrawal, the biochemistry, immunological and virulogical data in each period, YMDD mutation, pre-C mutation and prognosis after relapse were recorded., Results: The periods of treatment of 32.6% of patients in this group were shorter than 52 weeks. The HBV DNA of 93.6% of patients turned negative at the time of lamivudine withdrawal and returned to positive in all of those patients when relapsed; of the 6.4% of patients who did not turn negative at the time of lamivudine withdrawal, their HBV DNA was elevated more than 2 log when relapsed. A majority of patients (71.7%) did not ask their physicians when they decided to withdraw from lamivudine. There were 61.5% of the other patients who withdrew from lamivudine on the advice of physicians but they were not followed up. The state of their illness in 71.7% (33/46) patients was more severe than before their lamivudine therapy. In these cases, the YMDD mutation was detected in 78.8% of patients (chi2 = 23.23, P<0.01), and pre-C mutation was detected in 84.8% of patients (chi2 = 21.04, P<0.01), higher than that in the cases with aggrevation of their illnesses before the lamivudine therapy. The median time between lamivudine withdrawal and relapse was 12 weeks; it was negatively correlated with ALT (r = 0.32, P<0.05) detected at the time of lamivudine withdrawal. The severity of the illness at the time of relapse was related with age (r = 0.40, P<0.01) and YMDD mutation (r = 0.31, P<0.05). The prognosis was related with the age of the patient (r = 0.49, P<0.01), the diagnosis (r = 0.39, P<0.01), TBil (r = 0.46, P<0.05) and ALT (r = 0.32, P<0.05) detected before lamivudine therapy, HBeAg became negative when lamivudine was withdrawn (r = 0.31, P<0.05), TBil (r = 0.55, P<0.01) and PTA (r = 0.57, P<0.01) detected when relapsed., Conclusion: A majority of patients did not follow the lamivudine treatment recommendation of the experts group on lamivudine clinical practice in China. The major reason for relapse perhaps was revived HBV DNA multiplication, which induces damage of hepatocytes after lamivudine withdrawal. The YMDD mutation and/or pre-C mutation may be one of the factors that aggravate the damage of hepatocytes during the relapse. The factors including age of the patient, diagnosis before the treatment, TBil and ALT detected before lamivudine therapy, HBeAg turning to negative when lamivudine is withdrawn, and TBil and PTA detected during relapse may all impact the prognosis.