Harnessing the lysosome-dependent antitumor activity of phenothiazines in human small cell lung cancer.

Phenothiazines are a family of heterocyclic compounds whose clinical utility includes treatment of psychiatric disorders as well as chemotherapy-induced emesis. Various studies have demonstrated that these compounds possess cytotoxic activities in tumor cell lines of different origin. However, there is considerable confusion regarding the molecular basis of phenothiazine-induced cell death. Lung cancer (LC) remains one of the most prevalent and deadly malignancies worldwide despite considerable efforts in the development of treatment strategies, especially new targeted therapies. In this work, we evaluated the potential utility of phenothiazines in human LC. We show that phenothiazines as single treatment decreased cell viability and induced cell death preferentially in small cell lung carcinoma (SCLC) over non small cell lung carcinoma (NSCLC) cell lines. Sensitivity to phenothiazines was not correlated with induction of apoptosis but due to phenothiazine-induced lysosomal dysfunction. Interestingly, the higher susceptibility of SCLC cells to phenothiazine-induced cell death correlated with an intrinsically lower buffer capacity in response to disruption of lysosomal homeostasis. Importantly, this effect in SCLC occurred despite mutation in p53 and was not influenced by intrinsic sensitivity/resistance toward conventional chemotherapeutic agents. Our data thus uncovered a novel context-dependent activity of phenothiazines in SCLC and suggest that phenothiazines could be considered as a treatment regimen of this disease, however, extended cell line analyses as well as in vivo studies are needed to make such conclusion.

Resistance to DNA-damaging treatment in non-small cell lung cancer tumor-initiating cells involves reduced DNA-PK/ATM activation and diminished cell cycle arrest.

Increasing evidence suggests that tumor-initiating cells (TICs), also called cancer stem cells, are partly responsible for resistance to DNA-damaging treatment. Here we addressed if such a phenotype may contribute to radio- and cisplatin resistance in non-small cell lung cancer (NSCLC). We showed that four out of eight NSCLC cell lines (H125, A549, H1299 and H23) possess sphere-forming capacity when cultured in stem cell media and three of these display elevated levels of CD133. Indeed, sphere-forming NSCLC cells, hereafter called TICs, showed a reduced apoptotic response and increased survival after irradiation (IR), as compared with the corresponding bulk cell population. Decreased cytotoxicity and apoptotic signaling manifested by diminished poly (ADP-ribose) polymerase (PARP) cleavage and caspase 3 activity was also evident in TICs after cisplatin treatment. Neither radiation nor cisplatin resistance was due to quiescence as H125 TICs proliferated at a rate comparable to bulk cells. However, TICs displayed less pronounced G2 cell cycle arrest and S/G2-phase block after IR and cisplatin, respectively. Additionally, we confirmed a cisplatin-refractory phenotype of H125 TICs in vivo in a mouse xenograft model. We further examined TICs for altered expression or activation of DNA damage repair proteins as a way to explain their increased radio- and/or chemotherapy resistance. Indeed, we found that TICs exhibited increased basal γH2AX (H2A histone family, member X) expression and diminished DNA damage-induced phosphorylation of DNA-dependent protein kinase (DNA-PK), ataxia telangiectasia-mutated (ATM), Krüppel-associated protein 1 (KAP1) and monoubiquitination of Fanconi anemia, complementation group D2 (FANCD2). As a proof of principle, ATM inhibition in bulk cells increased their cisplatin resistance, as demonstrated by reduced PARP cleavage. In conclusion, we show that reduced apoptotic response, altered DNA repair signaling and cell cycle perturbations in NSCLC TICs are possible factors contributing to their therapy resistance, which may be exploited for DNA damage-sensitizing purposes.

Virtual reality simulator training equals mechanical robotic training in improving robot-assisted basic suturing skills.

BACKGROUND: This study aimed to investigate the effect of a virtual reality simulator on the learning of basic robotic suturing skills. METHODS: Two randomized groups of students underwent a controlled training program. Both groups completed an identical test before and after training. The increase in the number of stitches placed during the pretest and posttest was used as an objective measure of the training effect. To evaluate the subjective feeling of understanding and mastering, the students indicated this on a visual analog scale. RESULTS: Both groups showed a significant increase in the number of stitches placed during the posttest, and an increase in subjective feeling of understanding and mastering. The increase did not differ between the groups, indicating that the virtual reality simulator equaled the mechanical trainer in training of robotic suturing technique. CONCLUSIONS: Training in basic robot-assisted suturing skills using a virtual reality simulator without additional training equaled training using a mechanical simulator.

Graft control by transit time flow measurement and intraoperative angiography in coronary artery bypass surgery.

BACKGROUND: The aim of this study was to compare the relationship between intraoperative transit time flow measurements and angiographic findings with long-term graft patency in 72 patients who underwent coronary artery bypass surgery. METHODS: Transit time flow measurements with recording of mean flow and pulsatility indexes were performed after completion of the anastomoses. Coronary angiography was performed on-table while the patients were still in general anesthesia, and then at follow-up three months and 12 months after surgery. Based on angiography, the grafts were graded as type A (fully patent), type B (having more than 50% diameter reduction), or type O (occluded). RESULTS: Of the 67 left internal mammary artery (LIMA) grafts, 51 (76%) were type A on-table, 14 (21%) were type B, and two (3%) were type O. Of the 57 saphenous vein grafts, 49 (86%) were type A, 7 (12%) were type B, and one (2%) was type O. For both LIMA and vein grafts, there were no differences in flow (p = 0.69 and 0.47, respectively) or pulsatility index (p = 0.79 and 0.83) between type A and B. There were also no differences in flow (p = 0.37 and 0.7) or pulsatility index (p = 0.37 and 0.24) between type B on-table that either normalized or persisted occluded at the follow-up. Transit time flow measurement failed to detect an occluded LIMA graft as shown by intraoperative angiography. CONCLUSIONS: Blood flow measurements performed intraoperatively could not identify significant lesions in arterial or vein grafts, and could not predict graft patency. We have become cautious in interpreting flow measurements alone and combine blood flow recordings with intraoperative angiography in the assessment of graft quality.

Are some memory deficits unique to lesions of the mammillary bodies?

The role of the mammillary bodies in human memory is still in debate. A recent model of human amnesia proposes similar functions for the mammillary bodies and the hippocampus. But the main evidence for this model comes from animal studies using the delayed non-matching to sample paradigm. We describe a patient who developed a severe memory impairment after surgical removal of a germinoma. Postsurgical high resolution MRI revealed bilaterally shrunken mammillary bodies and an infarct of the left mammillary body. There were no other relevant lesions. Neuropsychological testing showed mildly impaired frontal lobe functions (executive functions, working memory and word fluency), almost intact learning and recognition, but severely impaired free and delayed recall. Experimental investigations revealed a reduced but preserved release of proactive interference and a pronounced impairment of recency and source judgments. We conclude that the mammillary bodies do play a prominent role in human memory, although the role differs slightly from that of the hippocampus.

Bile acid synthesis in cultured human hepatocytes: support for an alternative biosynthetic pathway to cholic acid.

The biosynthesis of bile acids by primary cultures of normal human hepatocytes has been investigated. A general and sensitive method for the isolation and analysis of sterols and bile acids was used, based on anion exchange chromatography and gas chromatography-mass spectrometry (GC/MS). Following incubation for 5 days, 8 oxysterols and 8 C(27)- or C(24)-bile acids were identified in media and cells. Cholic and chenodeoxycholic acids conjugated with glycine or taurine were by far the major steroids found, accounting for 70% and 24% of the total, respectively, being consistent with bile acid synthesis in human liver. Small amounts of sulfated 3beta-hydroxy-5-cholenoic acid and 3beta,7alpha-dihydroxy-5beta-cholanoic acid were also detected. Nine steroids were potential bile acid precursors (2% of total), the major precursors being 7alpha, 12alpha-dihydroxy-3-oxo-4-cholenoic acid and its 5beta-reduced form. These 2 and 5 other intermediates formed a complete metabolic sequence from cholesterol to cholic acid (CA). This starts with 7alpha-hydroxylation of cholesterol, followed by oxidation to 7alpha-hydroxy-4-cholesten-3-one and 12alpha-hydroxylation. Notably, 27-hydroxylation of the product 7alpha, 12alpha-dihydroxy-4-cholesten-3-one and further oxidation and cleavage of the side chain precede A-ring reduction. A-Ring reduction may also occur before side-chain cleavage, but after 27-hydroxylation, yielding 3alpha,7alpha, 12alpha-trihydroxy-5beta-cholestanoic acid as an intermediate. The amounts of the intermediates increased in parallel to those of CA during 4 days of incubation. Suppressing 27-hydroxylation with cyclosporin A (CsA) resulted in a 10-fold accumulation of 7alpha, 12alpha-dihydroxy-4-cholesten-3-one and a decrease of the production of CA and its acidic precursors. These results suggest that the observed intermediates reflect an alternative biosynthetic pathway to CA, which may be quantitatively significant in the cells.

Bile acid synthesis in cultured human hepatoblastoma cells.

Biosynthetic pathways to bile acids have been studied in HepG2 cells, a well-differentiated human hepatoblastoma cell line. Cholesterol metabolites, in total 29, were isolated from culture media and cells by liquid-solid extraction and anion-exchange chromatography and were identified by gas-liquid chromatography-mass spectrometry. The production rates/concentrations of cholic acid (CA) and chenodeoxycholic acid (CDCA) in media from control cells were 71 and 74 ng/10(7) cells/h, respectively. Major bile acid precursors were 3 alpha, 7 alpha, 12 alpha-trihydroxy-5 beta-cholestanoic acid (THCA), 7 alpha, 12 alpha-dihydroxy-3-oxo-4-cholestenoic acid, 7 alpha-hydroxy-3-oxo-4-cholenoic acid, and 7 alpha, 12 alpha-dihydroxy-3-oxo-5 beta-cholanoic acid, their concentrations being 60, 30, 23, and 10 ng/10(7) cells/h, respectively. These and nine other isolated intermediates formed essentially complete metabolic sequences from cholesterol to CA and CDCA. The remaining steroids were metabolites of the intermediates or autooxidation products of cholesterol. These findings and the observed effect of dexamethasone on production rates suggest that in HepG2 cells the major biosynthetic pathways to primary bile acids start with 7 alpha-hydroxylation of cholesterol and oxidation to 7 alpha-hydroxy-4-cholesten-3-one followed by hydroxylation at either the 26 or 12 alpha position. CDCA is formed by the sequence of 26-hydroxylation, oxidation, and degradation of the side chain and A-ring reduction. CA is formed by the sequence of 12 alpha-hydroxylation, 26-hydroxylation, oxidation, and degradation of the side chain and reduction of the A-ring. An alternative pathway to CA included A-ring reduction of the intermediate 7 alpha, 12 alpha-dihydroxy-3-oxo-4-cholestenoic acid to form THCA prior to side chain cleavage. These pathways are not limited to HepG2 cells but may also be important in humans.

Ethanol has an acute effect on bile acid biosynthesis in man.

A single dose of ethanol, 0.4 g/kg body weight, was found to give a 5-15 fold increase of the plasma concentrations of 7 alpha-hydroxy-cholesterol and 7 alpha-hydroxy-4-cholesten-3-one in humans. The rise was maximal 4 h after ethanol ingestion, was dose-dependent and was not seen in a cholecystectomized subject. The effect was selective for these and some other 7 alpha-hydroxylated C27-intermediates in bile acid biosynthesis. The changes are compatible with an acute stimulation of cholesterol 7 alpha-hydroxylase possibly due to an ethanol-induced inhibition of gallbladder contraction resulting in an interruption of the enterohepatic circulation of bile acids. The effect is of interest in relation to the influence of ethanol consumption on cardiovascular and gallstone diseases.

Concentrations of cholestenoic acids in plasma from patients with liver disease.

The concentrations of 3 beta-hydroxy-5-cholestenoic acid, 3 beta,7 alpha-dihydroxy-5-cholestenoic acid, and 7 alpha-hydroxy-3-oxo-4-cholestenoic acid were determined in plasma from patients with different liver diseases and compared with those of unconjugated and conjugated C24 bile acids. The levels of the cholestenoic acids were similar in patients with extrahepatic cholestasis and in controls (median concentration 153 and 162 ng/ml, respectively), whereas significantly elevated levels were found in plasma from patients with primary biliary cirrhosis (median concentration 298 ng/ml) and alcoholic liver cirrhosis (median concentration 262 ng/ml). As expected, conjugated C24 bile acids were elevated in most patients whereas the corresponding unconjugated compounds were low in cholestasis and elevated in alcoholic liver cirrhosis. The levels of the individual C27 acids were usually positively correlated to each other and also to the levels of conjugated C24 bile acids in plasma from patients with liver cirrhosis. In contrast, there was no correlation between the levels of C27 acids and conjugated bile acids in patients with extrahepatic cholestasis. The levels of unconjugated C24 bile acids were not correlated to C27 acids or conjugated bile acids in any of the groups. The results indicate that there is a close metabolic relationship between the individual C27 acids, that they do not participate in an enterohepatic circulation, and that the liver is important for their elimination/metabolism.

Concentrations of cholestenoic acids in plasma from patients with reduced intestinal reabsorption of bile acids.

The concentrations of 3 beta-hydroxy-5-cholestenoic acid, 3 beta,7 alpha-dihydroxy-5-cholestenoic acid, and 7 alpha-hydroxy-3-oxo-4-cholestenoic acid were determined in plasma from patients treated with cholestyramine or subjected to resection of the ileum or colon. The values were compared with those for conjugated and unconjugated C24 bile acids. Patients with an intact ileum but without colon had normal levels of cholestenoic acids. Patients treated with cholestyramine or with ileal resection had elevated levels of 7 alpha-hydroxy-3-oxo-4-cholestenoic acid (median values 189 and 233 ng/ml, respectively, compared to 85 ng/ml in controls). The levels of the other two C27 acids were normal in cholestyramine-treated and low in ileoresected patients and were positively correlated to each other but not to the 3-oxo-delta 4 acid. There were no consistent correlations between the levels of C27 acids and those of conjugated or unconjugated C24 bile acids. The results indicate an increased formation of 7 alpha-hydroxy-3-oxo-4-cholestenoic acid in subjects having a stimulated activity of cholesterol 7 alpha-hydroxylase.

Occurrence of 3 beta-hydroxy-5-cholestenoic acid, 3 beta,7 alpha-dihydroxy-5-cholestenoic acid, and 7 alpha-hydroxy-3-oxo-4-cholestenoic acid as normal constituents in human blood.

Three unconjugated C27 bile acids were found in plasma from healthy humans. They were isolated by liquid-solid extraction and anion-exchange chromatography and were identified by gas-liquid chromatography-mass spectrometry, microchemical reactions, and ultraviolet spectroscopy as 3 beta-hydroxy-5-cholestenoic, 3 beta,7 alpha-dihydroxy-5-cholestenoic, and 7 alpha-hydroxy-3-oxo-4-cholestenoic acids. Their levels often exceeded those of the unconjugated C24 bile acids and the variations between individuals were smaller than for the C24 acids. The concentrations in plasma from 11 healthy subjects were 67.2 +/- 27.9 ng/ml (mean +/- SD) for 3 beta-hydroxy-5-cholestenoic acid, 38.9 +/- 25.6 ng/ml for 3 beta,7 alpha-dihydroxy-5-cholestenoic acid, and 81.7 +/- 27.9 ng/ml for 7 alpha-hydroxy-3-oxo-4-cholestenoic acid. The levels of the individual acids were positively correlated to each other and not to the levels of the C24 acids. The cholestenoic acids were below the detection limit (20-50 ng/ml) in bile and C27 bile acids present in bile were not detected in plasma.