Haprolid Inhibits Tumor Growth of Hepatocellular Carcinoma through Rb/E2F and Akt/mTOR Inhibition.

BACKGROUND: Hepatocellular carcinoma (HCC) represents a major health burden with limited curative treatment options. There is a substantial unmet need to develop innovative approaches to impact the progression of advanced HCC. Haprolid is a novel natural component isolated from myxobacteria. Haprolid has been reported as a potent selective cytotoxin against a panel of tumor cells in recent studies including HCC cells. The aims of this study are to evaluate the antitumor effect of haprolid in HCC and to understand its underlying molecular mechanisms. METHODS: The efficacy of haprolid was evaluated in human HCC cell lines (Huh-7, Hep3B and HepG2) and xenograft tumors (NMRI-Foxn1nu mice with injection of Hep3B cells). Cytotoxic activity of haprolid was determined by the WST-1 and crystal violet assay. Wound healing, transwell and tumorsphere assays were performed to investigate migration and invasion of HCC cells. Apoptosis and cell-cycle distribution were measured by flow cytometry. The effects of haprolid on the Rb/E2F and Akt/mTOR pathway were examined by immunoblotting and immunohistochemistry. RESULTS: haprolid treatment significantly inhibited cell proliferation, migration and invasion in vitro. The epithelial-mesenchymal transition (EMT) was impaired by haprolid treatment and the expression level of N-cadherin, vimentin and Snail was downregulated. Moreover, growth of HCC cells in vitro was suppressed by inhibition of G1/S transition, and partially by induction of apoptosis. The drug induced downregulation of cell cycle regulatory proteins cyclin A, cyclin B and CDK2 and induced upregulation of p21 and p27. Further evidence showed that these effects of haprolid were associated with Rb/E2F downregulation and Akt/mTOR inhibition. Finally, in vivo nude mice experiments demonstrated significant inhibition of tumor growth upon haprolid treatment. CONCLUSION: Our results show that haprolid inhibits the growth of HCC through dual inhibition of Rb/E2F and Akt/mTOR pathways. Therefore, haprolid might be considered as a new and promising candidate for the palliative therapy of HCC.

Two patients with intestinal failure requiring home parenteral nutrition, a NOD2 mutation and tuberculous lymphadenitis.

BACKGROUND: Mutations in the NOD2 gene are a significant risk factor to acquire intestinal failure requiring home parenteral nutrition. Tuberculous lymphadenitis is the main manifestation of extrapulmonary tuberculosis. Defects in the innate immunity, including NOD2 mutations, may increase the risk for acquiring infections caused by M. tuberculosis. An association of intestinal failure, mutations in the NOD2 gene and tuberculous lymphadenitis has not been described before. CASE PRESENTATION: We report of two patients with intestinal failure secondary to mesenteric ischemia. Both patients presented with fever and weight loss while receiving long term home parenteral nutrition. Both of them were found to have mutations in the NOD2 gene. Catheter related infections were ruled out. FDG-PET-CT scans initially obtained in search for another infectious focus that would explain the symptoms unexpectedly showed high FDG uptake in mediastinal lymph nodes. Direct or indirect evidence proved or was highly suggestive for tuberculous lymphadenitis. Intravenous tuberculostatic therapy was started and led to a reversal of symptoms and to resolution of the lesions by FDG-PET-CT. CONCLUSION: Mutations in the NOD2 gene may put patients both at an increased risk for acquiring M. tuberculosis infections as well as at an increased risk of intestinal failure after extensive intestinal resection. Thus we suggest to specifically include reactivated and opportunistic infections in the differential diagnosis of suspected catheter related infection in patients with intestinal failure who carry mutations in their NOD2 gene.

NOD2 mutations are associated with the development of intestinal failure in the absence of Crohn's disease.

BACKGROUND & AIMS: Short bowel syndrome (SBS) and intestinal failure (IF) are multi-factorial conditions which in adults result from extensive intestinal resection. NOD2 is an intracellular pattern recognition receptor associated with CD. An unexpected high frequency of NOD2 mutations has been found in patients undergoing intestinal transplantation (35%). The role of NOD2 in a cohort with SBS/IF not specifically requiring intestinal transplantation has not been studied yet. METHODS: The course of 85 patients with non-malignant SBS/IF was characterized. The major NOD2 mutations, as well as ATG16L1 and IL23R were determined. The allele frequencies were compared to the published frequencies of CD patients and controls. RESULTS: In non-CD patients (72%) allele frequencies of NOD2 mutations were statistically more frequent than in controls (14% vs 6%, p = 0.006). In CD patients (28%) allele frequencies were not different between SBS and controls (29% vs 22%, p = 0.23). NOD2 mutations were neither associated with parameters potentially heralding the need for transplantation nor with an earlier time to the indication for intestinal transplantation. CONCLUSIONS: NOD2 mutations are associated with the development of SBS/IF in the absence of CD, but not with specific complications. NOD2 mutations may increase the risk for more extensive intestinal resection or may impair intestinal adaptation.

Quorum sensing in the probiotic bacterium Escherichia coli Nissle 1917 (Mutaflor) - evidence that furanosyl borate diester (AI-2) is influencing the cytokine expression in the DSS colitis mouse model.

BACKGROUND: "Quorum sensing" (QS) is the phenomenon which allows single bacterial cells to measure the concentration of bacterial signal molecules. Two principle different QS systems are known, the Autoinducer 1 system (AI-1) for the intraspecies communication using different Acyl-homoserine lactones (AHL) and AI-2 for the interspecies communication. Aim of this study was to investigate QS of Escherichia coli Nissle 1917 (Mutaflor). RESULTS: While E. coli Nissle is producing AI-2 in a density dependent manner, no AI-1 was produced. To study the effect of AI-2 in the DSS (dextran sulphate sodium) induced mouse model of acute colitis, we silenced the corresponding gene luxS by intron insertion. The mutant bacterium E. coli Nissle::luxS was equally effective in colonizing the colon and the mutation turned out to be 100% stable during the course of the experiment. Isolating RNA from the colon mucosa and performing semiquantitative RT PCR, we were able to show that the expression of the pro-inflammatory cytokine IFN-y was suppressed in mice being infected with the E. coli Nissle wild type. Mice infected with the E. coli Nissle::luxS mutant showed a suppressed expression of IL-10 compared to uninfected mice, while the expression of the pro-inflammatory cytokines IL-6 and TNF-α was higher in these mice. The expression of mBD-1 was suppressed in mice being infected with the mutant in comparison to the mice not infected or infected with the wild type. No differences were seen in the histological examination of the colon sections in the different groups of mice. CONCLUSIONS: E. coli Nissle is producing AI-2 molecules, which are influencing the expression of cytokines in the mucosa of the colon in the DSS mice. However, if QS has a direct influence on the probiotic properties of E. coli Nissle remains to be elucidated.

Intestinal anion exchanger down-regulated in adenoma (DRA) is inhibited by intracellular calcium.

The Na/H exchanger 3 (NHE3) and the Cl/HCO(3) exchanger down-regulated in adenoma (DRA) together facilitate intestinal electroneutral NaCl absorption. Elevated Ca(2+)(i) inhibits NHE3 through mechanisms involving the PDZ domain proteins NHE3 kinase A regulatory protein (E3KARP) or PDZ kidney 1 (PDZK1). DRA also possesses a PDZ-binding motif, but the roles of interactions with E3KARP or PDZK1 and Ca(2+)(i) in DRA regulation are unknown. Wild type DRA and a mutant lacking the PDZ interaction motif (DRA-ETKFminus) were expressed constitutively in human embryonic kidney (HEK) and inducibly in Caco-2/BBE cells. DRA-mediated Cl/HCO(3) exchange was measured as intracellular pH changes. Ca(2+)(i) was assessed fluorometrically. DRA was induced 8-16-fold and was delivered to the apical surface of polarized Caco-2 cells. Putative anion transporter 1 and cystic fibrosis transmembrane regulator did not contribute to Cl/HCO(3) exchange in transfected Caco-2 cells. The calcium ionophore 4Br-A23187 inhibited DRA and DRA-ETKFminus in HEK cells, but only full-length DRA was inhibited in Caco-2 cells. In contrast, 100 microm UTP, which increased Ca(2+)(i), inhibited full-length DRA but not DRA-ETKFminus in Caco-2 and HEK cells. In HEK cells, which express little PDZK1, additional transfection of PDZK1 was required for UTP to inhibit DRA. As HEK cells do not express cystic fibrosis transmembrane regulator or NHE3, the data indicate that Ca(2+)(i)-dependent DRA inhibition is not because of modulation of other transport activities. In polarized epithelium, this inhibition requires interaction of DRA with PDZK1. Together with data from PDZK1(-/-) mice, these data underscore the prominent role of PDZK1 in Ca(2+)(i)-mediated inhibition of colonic NaCl absorption.

Prognostic implications of BAX protein expression and microsatellite instability in all non-metastatic stages of primary colon cancer treated by surgery alone.

PURPOSE: This study examined whether the apoptosis-related protein, BAX, or the microsatellite-instability phenotype provide prognostic information in patients with resected colon cancer. METHODS: A total of 371 stage I-III patients that previously underwent radical surgery were included (mean follow-up 51.8 months). BAX expression was examined by immunohistochemical staining; high-frequency microsatellite instability (MSI+) was determined by assessing the specific marker, BAT26, using single-strand conformation polymorphism (SSCP)-based analysis. RESULTS: High BAX expression was found in 66.4% of patients. MSI+ tumors were observed in 14.8% of 344 patients. Univariate analysis showed that unlike MSI, low BAX expression was significantly correlated with poor disease-specific overall survival (OS) in stages I-III (p = 0.04). Multivariate subgroup analyses revealed that unlike MSI, low BAX was an independent predictor for OS in stage II (p = 0.009); however, in stages I or III, BAX or MSI were not independent predictors of OS. CONCLUSIONS: In stage II colon cancer treated with surgery alone, BAX protein expression may be a predictor for prognosis.

Epigenetic combination therapy as a tumor-selective treatment approach for hepatocellular carcinoma.

BACKGROUND: Innovative epigenetic therapeutics comprise histone deacetylase inhibitors (HDAC-I) and demethylating agents (DA). It was recently found that HDAC-I compounds exhibit profound therapeutic activities against hepatocellular carcinoma (HCC). A comprehensive preclinical investigation was performed on the potential of a combined HDAC-I/DA epigenetic regimen for the highly chemotherapy-resistant HCC entity. METHODS: Human HCC-derived cell lines or primary human hepatocytes (PHH) were treated with HDAC-I compound suberoylanilide hydroxamic acid (SAHA) or DA compound 5-aza-2'-deoxycytidine (5-aza-dC) or both and examined for cellular damage, proliferation, histone acetylation pattern, and DNA methylation. In vivo activities were investigated in a xenograft hepatoma model. RESULTS: Monotherapeutic application of SAHA or 5-aza-dC was found to induce substantial antiproliferative effects in HCC-derived cells, strongly enhanced by combined SAHA and 5-aza-dC treatment. PHH from different human donors did not exhibit any relevant cellular damage even when applying high doses of the combination regimen, whereas HCC-derived cell lines showed a dose-dependent damage. In vivo testing demonstrated a statistical significant inhibition of hepatoma cell growth for the combined treatment regime. CONCLUSIONS: Because the combined HDAC-I/DA epigenetic approach was found to produce significant antitumor effects in HCC model systems and did not impair cellular integrity of untransformed hepatocytes, this combination therapy is now considered for further investigation in clinical trials.

Low BAX protein expression correlates with disease recurrence in preoperatively irradiated rectal carcinoma.

PURPOSE: To determine the prognostic impact of BAX in correlation to its upstream effector p53 as well as clinicopathologic variables and patient outcome in preoperatively irradiated rectal carcinoma. METHODS AND MATERIALS: We investigated 92 rectal carcinoma patients treated by preoperative radiotherapy to a total dose of 30 Gy followed by surgery. Median follow-up was 71 months. Immunohistochemistry was performed on paraffin sections of pretreatment biopsy samples for BAX protein. Also, we considered the previously determined p53 expression data from this cohort. RESULTS: BAX protein expression was classified as high and low in 63 (68.5%) and 29 (31.5%) tumors, respectively. Unlike clinicopathologic variables, high BAX expression was significantly associated with improved disease-free survival by univariate analysis (p = 0.048). Moreover, in multivariate analyses, high BAX expression was an independent prognostic indicator for both improved local recurrence-free interval and improved disease-free survival (p = 0.03 and 0.047, respectively). Concerning the p53/BAX pathway, subgroup analysis yielded no association between p53 immunonegative/BAX high vs. p53 immunopositive/BAX low expressing tumors with regard to overall, disease-free, or local recurrence-free survival in either univariate (p = 0.88, 0.54, and 0.16, respectively) or multivariate analysis. CONCLUSIONS: This study demonstrates that BAX protein expression might help to predict disease recurrence in preoperatively irradiated rectal carcinoma, whereas determination of p53, the proposed upstream regulator of BAX-induced apoptosis, did not provide additional prognostic information.

Promoter methylation of INK4a/ARF as detected in bile-significance for the differential diagnosis in biliary disease.

PURPOSE: There is a need to enhance endobiliary cytotechniques by molecular marker lesions. This is of special significance for patients with primary sclerosing cholangitis, a disease predisposing for the development of cholangiocarcinoma. The INK4a/ADP ribosylation factor (ARF) locus encodes two tumor suppressor genes: p16INK4a and p14ARF. p16INK4a has been shown to be of major significance in cholangiocarcinoma. EXPERIMENTAL DESIGN: In an effort to evaluate the potential diagnostic role of p16INK4a and p14ARF promoter methylation in biliary disease, endoscopical obtained bile specimens of 71 patients were analyzed (26 choledocholithiasis, 6 with normal results, 23 bile duct carcinoma, 5 gall bladder carcinoma). Eleven patients with primary sclerosing cholangitis were enrolled. RESULTS: Merely 6% of specimens (2 of 32) obtained from patients without evidence for malignant biliary disease but 53.5% of malignancies (15 of 28) showed p16 promoter methylation (p14: 3 and 46.2%, respectively). The concordance of methylation rates detected in either bile or tissue specimens was high. In primary sclerosing cholangitis, a similar prevalence of methylation was detected as in malignant disease. CONCLUSIONS: This study demonstrates: (a) a high frequency and specificity of INK4a/ARF methylation in malignant biliary disease compared with mere cholangitis; and (b) the capability to detect these alterations reliably in endoscopically obtained bile. Thus, INK4a/ARF's promoter methylation status represents a candidate marker for the endoscopic diagnosis of biliary disease.