Silencing of Twist1 sensitizes NSCLC cells to cisplatin via AMPK-activated mTOR inhibition.

Twist1 is highly expressed in primary and metastatic non-small cell lung cancer (NSCLC), and thus acts as a critical target for lung cancer chemotherapy. In the current study, we investigated the underlying mechanism initiated by silencing of Twist1 that sensitizes NSCLC cells to cisplatin. Silencing of Twist1 triggered ATP depletion, leading to AMP-activated protein kinase (AMPK)-activated mammalian target of rapamycin (mTOR) inhibition in NSCLC cells. AMPK-induced mTOR inhibition, in turn, resulted in downregulation of ribosome protein S6 kinase 1 (S6K1) activity. Downregulation of mTOR/S6K1 reduced Mcl-1 protein expression, consequently promoting sensitization to cisplatin. Overexpression of Mcl-1 reduced PARP cleavage induced by cisplatin and Twist1 siRNA, suggesting that this sensitization is controlled through Mcl-1 expression. Interestingly, cells treated with Twist1 siRNA displayed upregulation of p21(Waf1/CIP1), and suppression of p21(Waf1/CIP1) with specific siRNA further enhanced the cell death response to cisplatin/Twist1 siRNA. In conclusion, silencing of Twist1 sensitizes lung cancer cells to cisplatin via stimulating AMPK-induced mTOR inhibition, leading to a reduction in Mcl-1 protein. To our knowledge, this is the first report to provide a rationale for the implication of cross-linking between Twist1 and mTOR signaling in resistance of NSCLC to anticancer drugs.

TXNIP potentiates Redd1-induced mTOR suppression through stabilization of Redd1.

The mammalian target of rapamycin (mTOR) is a highly conserved serine-threonine kinase activated in response to growth factors and nutrients. Because of frequent dysregulation of the mTOR signaling pathway in diverse human cancers, this kinase is a key therapeutic target. Redd1 is a negative regulator of mTOR, mediating dissociation of 14-3-3 from tuberous sclerosis complex (TSC)2, which allows formation of a TSC-TSC2 complex. In the present study, we identify TXNIP that inhibits mTOR activity by binding to and stabilizing Redd1 protein. Redd1 and TXNIP expression was induced by a synthetic glucose analog, 2-deoxyglucose (2-DG). Moreover, Redd1 expression in response to 2-DG was regulated by activating transcription factor 4 (ATF4). Overexpression of TXNIP was associated with reduced mTOR activity mediated by an increase in Redd1 level, whereas knockdown of TXNIP using small interfering RNA resulted in recovery of mTOR activity via downregulation of Redd1 during treatment with 2-DG. Interestingly, Redd1 was additionally stabilized via interactions with N-terminal-truncated TXNIP, leading to suppression of mTOR activity. Our results collectively demonstrate that TXNIP stabilizes Redd1 protein induced by ATF4 in response to 2-DG, resulting in potentiation of mTOR suppression. To the best of our knowledge, this is the first study to identify TXNIP as a novel member of the mTOR upstream that acts as a negative regulator in response to stress signals.

Growth and photosynthetic responses of two pine species (Pinus koraiensis and Pinus rigida) in a polluted industrial region in Korea.

We investigated the effects of pollutants on two pine species (Pinus koraiensis and Pinus rigida) in an industrial region in Korea, using a physiological approach. The concentrations of fluorine (F) and chlorine (Cl) in the atmosphere, in precipitation and soil water at the damaged site were all significantly higher than at a control site. Moreover, the concentrations of F, Cl and Mn in pine needles were significantly higher, and essential elements and chlorophyll in needles were significantly lower at the damaged site than at the control site. The photosynthetic capacities, shoot length and survival statistics of needles of the two pines were all significantly reduced at the damaged site compared to the control site, especially P. rigida. Based on our comparison of photosynthetic responses and the concentrations of F, Cl and Mn in needles of the two pine species, P. koraiensis is more resistant to excess Mn in its needles than P. rigida.

Colonic motility abnormality in patients with irritable bowel syndrome exhibiting abdominal pain and diarrhea.

OBJECTIVES: Although colon dysmotility is recognized as a pathophysiological factor in irritable bowel syndrome (IBS), it has not been characterized. We have investigated motility patterns in IBS patients with abdominal pain and frequent defecation or diarrhea and in healthy volunteers. METHODS: A recording catheter that had six polyvinyl tubes with infusion ports was placed in the transverse, descending, and sigmoid colon under fluoroscopy. After 2-h basal recordings, motility responses to cholecystokinin octapeptide (CCK-8) and a meal were studied for 3 h. The motility index (MI) and number of high amplitude propagating contractions (HAPCs) in 10 IBS patients were compared with those of 10 controls. HAPCs were correlated with abdominal pain, and colon transit time using radio-opaque markers was determined. Using human colon muscle strips, the effect of CCK-8 on muscle contractions was also studied. RESULTS: The MI and mean number and peak amplitude of HAPCs in IBS patients were significantly greater than in controls. These abnormalities paralleled markedly shortened colonic transit time. Abdominal pain coincided with >90% of HAPCs. Dose-dependent muscle contraction by CCK-8 was profoundly suppressed both by loxiglumide and atropine. CONCLUSIONS: The dysmotility in this subset of IBS patients was characterized by significantly increased occurrences of powerful HAPCs that paralleled rapid colon transit and were accompanied by abdominal pain. Thus, it is suggested that this powerful contraction is one of the causes of abdominal pain. The action of CCK-8 seems to be mediated via the colon enteric nervous system.

Prevalence of Helicobacter pylori in peptic ulcer patients in greater Rochester, NY: is empirical triple therapy justified?

OBJECTIVES: Among patients with peptic ulcer disease, the prevalence of Helicobacter pylori has been reported to range from 80% to 90%. Thus empirical cost-effective therapy has been suggested. We surveyed patients with peptic ulcer disease in Rochester, NY. METHODS: From two teaching hospitals all patients who had duodenal ulcers (DU) and/or gastric ulcers (GU) on esophagogastroduodenoscopy (EGD) with antral biopsy for histology for H. pylori and for rapid urease (CLO) test were included in the study. We examined a total of 160 patients with DU and 145 patients with GU, age range 18-92 yr, obtaining clinical data, race, medication profile, and history of use of nonsteroidal antiinflammatory drugs (NSAIDs). An ulcer was defined if the lesion with loss of mucosal integrity was > or = 0.5 cm, with apparent depth. H. pylori was considered present if CLO test and/or histology were positive for H. pylori. To confirm the reliability of nonuse of NSAIDs, we randomly checked blood samples of 90 such patients from the ambulatory clinic for the presence of salicylates. To identify the sensitivity of the CLO test, we performed a serology test for H. pylori antibody in 100 subjects to compare the CLO test results. Also, 500 CLO test results were compared to the histology results for H. pylori. RESULTS: Among 160 DU patients, 16 were NSAID users with negative H. pylori and excluded from the prevalence study. Of the remaining 144 patients with DU, H. pylori was present in 88 patients (61%). When these data were analyzed according to race, H. pylori was present in 54 (52%) of 104 whites compared to 34 of 40 (85%) nonwhites (blacks, Hispanics, Asians) (p < 0.01). Among 145 GU patients 18 were NSAID users with negative H. pylori and excluded from the prevalence analysis. Of the remaining 127 patients with GU, H. pylori was present in 87 patients (61%). Among them, H. pylori was present in 46 of 87 (53%) whites, whereas 31 of 40 nonwhites (78%) were H. pylori-positive (p < 0.01). Antral histology and CLO test for H. pylori were in agreement in 92% of cases. Serology and CLO test for H. pylori were in agreement in 87% of cases. None of the randomly screened patients, including 16 ulcer patients with negative H. pylori, showed presence of salicylate in blood. CONCLUSION: In greater Rochester, NY, where the majority of our patients with EGD were whites, the prevalence of H. pylori among ulcer patients was lower compared to other regions, particularly among whites. This suggests that an additional causative factor or factors for peptic ulcers may be present. Hence, empirical antibiotic therapy of ulcer patients without confirming the presence of H. pylori may not be justified.

Inhibition of acid secretion by electrical acupuncture is mediated via beta-endorphin and somatostatin.

Electroacupuncture (EAP) was shown to inhibit basal gastric acid secretion in dogs and sham feeding-stimulated acid secretion in humans. However, its effect on a meal-stimulated acid secretion in dogs and the mechanisms involved remain unclear. In five dogs prepared with gastric cannulas, gastric acid secretion was determined by a dye-dilution technique for 60 min after intragastric administration of 200 ml of 4% mixed amino acid meal in six different experiments: study 1, no acupuncture; study 2, sham acupuncture (SAP); study 3, EAP; study 4, EAP plus naloxone; study 5, naloxone alone; and study 6, intravenous infusion of somatostatin (SS) and vasoactive intestinal peptide (VIP) at doses of 0.5 and 1.0 micrograms.kg-1.h-1, respectively. EAP was performed on three different points including Pishu, ZusanLi, and Neiguan. Biphasic electrical pulse (25-100 Hz, 12-16 mA) was applied continuously via needles for 75 min starting 15 min before meal. SAP on nonacupoints in hind- and forelegs was performed with the same electrical pulse. Plasma SS, VIP, beta-endorphin, and gastrin were determined by specific radioimmunoassays. EAP significantly inhibited acid secretion (75%; P < 0.01), which coincided with significant increases in plasma SS, VIP, and beta-endorphin and a significant decrease in plasma gastrin. Naloxone completely reversed EAP-induced inhibition of acid secretion and changes in plasma concentration of peptides. SAP also significantly suppressed acid output (30%; P < 0.05), with a modest but significant increase in plasma beta-endorphin. However, the inhibition by EAP on the acid output was significantly greater than that by SAP (P < 0.01). Furthermore, exogenous SS (0.5 microgram.kg-1.h-1) significantly inhibited acid output (78%), whereas VIP failed to inhibit gastric acid secretion. We conclude that, in dogs, EAP significantly inhibits meal-stimulated acid secretion. This acid inhibition is mediated by the release of beta-endorphin and somatostatin, and an endogenous opiate or opiates appear to play an important role in the release of SS, VIP, and beta-endorphin.

Roles of gut hormones in negative-feedback regulation of pancreatic exocrine secretion in humans.

BACKGROUND/AIMS: Secretin has been shown to mediate feedback control of pancreatic secretion of fluid and bicarbonate in rats, guinea pigs, and dogs. However, little is known about secretin in the feedback regulation in humans. We investigated the roles of secretin, cholecystokinin, neurotensin, and pancreatic polypeptide on feedback regulation of pancreatic secretion in 10 human volunteers. METHODS: A 5-lumen tube was positioned in the proximal jejunum of fasting subjects under fluoroscopy so that gastric juice via lumen 1 and duodenal contents via lumen 3 were collected separately in 15-minute samples while polyethylene glycol solution was infused into duodenum via lumen 2. An acidified (pH 2.0) 4.25% amino acid mixed with phenol red was infused into proximal jejunum via lumen 4, which was alternated with NaHCO3 (control solution) or trypsin (test solution) via lumen 5 intermittently every 15 minutes during separate test periods. RESULTS: Infusion of control solution significantly increased both bicarbonate (total change [delta], 7799 +/- 1400 mumol/h) and chymotrypsin (delta 5500 +/- 762 mumol/h) outputs and levels of all four plasma hormones. The test solution significantly inhibited both bicarbonate (delta 2999 +/- 700 mumol/h; P < 0.01) and chymotrypsin output (delta 1000 +/- 120 U/h, P < 0.01), which coincided with a significant suppression of plasma concentration of secretin and cholecystokinin but not pancreatic peptide and neurotensin. CONCLUSIONS: A negative-feedback regulation of pancreatic secretion of bicarbonate and enzyme occurs in humans and is mediated via both secretin and cholecystokinin.

Physiological role of cholecystokinin on gastric emptying and acid output in dogs.

We investigated the physiological role of cholecystokinin (CCK) on gastric emptying and acid secretion in seven conscious dogs with gastric cannulae. Two hundred milliliters of a 4% amino acid meal was given via the cannula, and both gastric emptying and acid output were measured concurrently using a dye-dilution technique. Gastric emptying of the liquid amino acid meal was exponential, and the acid output and plasma concentrations of CCK, gastrin, and somatostatin peaked within 30 min after the meal. Intravenous infusion of CCK-8 at 28 and 56 pmol/kg/hr but not 14 pmol/kg/hr increased plasma levels of the peptide and inhibited gastric emptying as well as acid output. Plasma gastrin was not affected significantly by the CCK infusion, whereas plasma somatostatin increased significantly in response to 56 pmol/kg/hr of CCK-8. Loxiglumide, 22 mumol/kg/hr, significantly enhanced gastric emptying and augmented acid output, as well as plasma gastrin response, whereas it abolished the postprandial rise in plasma somatostatin. We concluded that in dogs, CCK plays an important role in the physiologic regulation of postprandial gastric emptying of a liquid caloric meal and acid output. Its inhibitory effect on gastric acid secretion appears to be mediated, at least in part, by somatostatin.

Secretin: a physiological regulator of gastric emptying and acid output in dogs.

Secretin has been known to inhibit gastric acid secretion in several species. However, the physiological role of secretin on the postprandial acid output and gastric emptying in an intact stomach remains controversial. In the present study, we reinvestigated the role of secretin in physiological dose range and endogenous secretin on gastric acid secretion and emptying in the stomach without influencing intragastric luminal pH in dogs. In seven conscious dogs with gastric cannulas, a 4% amino acid meal was administered intragastrically, and three different doses of secretin and an antisecretin serum were infused intravenously in each dog on separate days. Gastric emptying and net acid output were measured using a dye dilution technique, and plasma secretin and gastrin were determined by specific radioimmunoassays. After the meal, gastric emptying was exponential: acid output peaked at 25 min, and plasma concentrations of gastrin and secretin peaked at 15 and 60 min, respectively. Intravenous infusion of secretin at 1.25, 2.5, and 5.0 pmol.kg-1.h-1 dose dependently increased plasma levels of the peptide and suppressed postprandial plasma gastrin response and gastric acid output and emptying of the meal. Immunoneutralization of circulating secretin with a rabbit antisecretin serum abolished the postprandial rise of plasma secretin and significantly increased plasma gastrin, and augmented gastric emptying as well as acid output. It is concluded that, in dogs, secretin plays a physiological role in the regulation of gastric emptying and acid output after a liquid amino acid meal and that these effects may be mediated in part by suppression of the release of gastrin.