Overexpression of polyphosphate polymerases and deletion of polyphosphate phosphatases shorten the replicative lifespan in yeast.

We previously found that overexpression of phosphate starvation-responsive genes by disrupting PHO80 led to a shortened replicative lifespan in yeast. To identify lifespan-related genes, we screened upregulated genes in the pho80Δ mutant and focused on the VTC genes, which encode the vacuolar polyphosphate (polyP) polymerase complex. VTC1/VTC2/VTC4 deletion restored the lifespan and intracellular polyP levels in pho80Δ. In the wild type, overexpression of VTC5 or a combination of the other VTCs caused high polyP accumulation and shortened lifespan. Similar phenotypes were caused by the deletion of polyP phosphatase genes-vacuolar PPN1 and cytosolic PPX1. The polyP-accumulating strains exhibited stress sensitivities. Thus, we demonstrated that polyP metabolic enzymes participate in replicative lifespan, and extreme polyP accumulation shortens the lifespan.

Secreted acid phosphatases maintain replicative lifespan via inositol polyphosphate metabolism in budding yeast.

Secreted acid phosphatases (APases) dephosphorylate extracellular organic phosphate compounds to supply inorganic phosphate (Pi) to maintain cellular functions. Here, we show that APases are necessary to maintain a normal replicative lifespan in Saccharomyces cerevisiae. Deletion of all four APase genes shortened the lifespan in yeast strains on synthetic media (irrespective of the concentrations of Pi in the media), but it did not affect the intracellular ortho- and polyphosphate levels. Deletion of inositol-pentakisphosphate 2-kinase (IPK1), which encodes inositol-pentakisphosphate 2-kinase, restored the lifespan in APase-null mutants, and IPK1 overexpression shortened the lifespan in wild-type strains. Overexpression of inositol hexakisphosphate (IP6 ) and heptakisphosphate kinases, KCS1 and VIP1, recovered the lifespan in APase-null mutants. Thus, yeast APases modulate the replicative lifespan, probably through dephosphorylation of intracellular IP6 .

Cyclin-dependent kinase Pho85p and its cyclins are involved in replicative lifespan through multiple pathways in yeast.

Lifespan is determined by genetic factors and influenced by environmental factors. Here, we find that the phosphate signal transduction (PHO) pathway is involved in the determination of replicative lifespan in budding yeast. Extracellular phosphate does not affect the lifespan. However, deletion of PHO80 (cyclin) and PHO85 (cyclin-dependent kinase) genes, that is, negative regulators of the PHO pathway, shortens the lifespan, which is restored by further deletion of PHO4 (transcriptional activator). Four of the other nine Pho85p cyclin genes are also required to maintain normal lifespan. The short-lived mutants show a metabolic profile that is similar to strains with normal lifespan. Thus, Pho85p kinase genetically determines replicative lifespan in combination with relevant cyclins. Our findings uncover novel cellular signals in longevity regulation.

Safety and efficacy of the sodium-glucose cotransporter 1 inhibitor mizagliflozin for functional constipation: a randomised, placebo-controlled, double-blind phase 2 trial.

BACKGROUND: Mizagliflozin is a novel oral sodium-glucose cotransporter 1 (SGLT1) inhibitor that increases luminal glucose and water. This study assessed the efficacy and safety of mizagliflozin in patients with functional constipation. METHODS: In this multicentre, randomised, double-blind phase 2 trial at 32 hospitals and community outpatient clinics in Japan, we enrolled patients with functional constipation or constipation-predominant irritable bowel syndrome, aged 20 years or older. Patients were randomly assigned (1:1:1), by use of an independent centralised registration system and dynamic allocation method, to receive mizagliflozin 5 mg, mizagliflozin 10 mg, or placebo, orally once daily for 4 weeks. Patients, investigators, staff, and the sponsor were blinded to the group assignments. The primary outcome was the change from baseline in the number of spontaneous bowel movements per week after 1 week. Efficacy analysis was done in all patients except those who deviated from good clinical practice, did not receive at least one dose of the study drug, withdrew before starting treatment, were ineligible, or for whom the primary outcome could not be assessed, and safety was assessed in all patients except those who deviated from good clinical practice, who did not receive the study drug, or who withdrew before receiving treatment. This trial is registered with ClinicalTrials.gov, number NCT02281630, and is completed. FINDINGS: Between Oct 15, 2014, and March 7, 2015, 258 patients with functional constipation were randomly assigned: 86 patients per group. Two patients from the placebo group and three from the 10 mg mizagliflozin group were excluded because the primary outcome could not be assessed, and one patient from the 5 mg mizagliflozin group was excluded for not receiving the study drug; therefore 84 patients in the placebo group, 85 in the 5 mg mizagliflozin group, and 83 in the 10 mg mizagliflozin group were included in the full analysis population. Mean change from baseline in the number of spontaneous bowel movements per week after 1 week with mizagliflozin 5 mg (3·85 [SD 3·96]) and mizagliflozin 10 mg (5·85 [6·01]) was significantly greater than those in the placebo group (1·80 [1·80]; p<0·0001 for both comparisons). The most common adverse events were nasopharyngitis (one [1%] of 86 patients in the placebo group, seven [8%] of 85 on mizagliflozin 5 mg, and five [6%] of 86 on mizagliflozin 10 mg), diarrhoea (none on placebo, four [5%] patients on mizagliflozin 5 mg, and eight [9%] on mizagliflozin 10 mg), and abdominal distention (three [3%] on placebo, four [5%] on mizagliflozin 5 mg, and seven [8%] on mizagliflozin 10 mg). Only diarrhoea and abdominal distention were deemed to be related to mizagliflozin treatment, whereas nasophanyngitis might not be related to mizagliflozin treatment, on the basis of clinical evaluation. INTERPRETATION: The SGLT1 inhibitor mizagliflozin showed favourable efficacy and tolerability at 5 mg and 10 mg doses in patients with functional constipation, providing a potential alternative therapy to available drugs. FUNDING: Kissei Pharmaceutical.

Involvement of cyclooxygenase-2 in gastric mucosal hypertrophy in gastrin transgenic mice.

Gastrin promotes gastric mucosal growth, and hypergastrinemia induces gastric mucosal hypertrophy. Recently, it has been reported that gastrin induces cyclooxygenase-2 (COX-2) in human gastric and colorectal cancer cell lines. However, whether COX-2 is involved in gastrin-induced gastric mucosal growth in vivo is unknown. We investigated the role of COX-2 in gastrin-induced gastric mucosal hypertrophy using gastrin transgenic mice. Hypergastrinemic mice [mice with mutated gastrin under the control of the beta-actin promoter (ACT-GAS mice)] received the COX-2 inhibitor celecoxib (0, 200, or 500 mg/kg of diet) from 5 wk of age and were killed at 16 or 24 wk. Some ACT-GAS mice received celecoxib from 16 wk and were killed at 24 wk. Eighty-week-old ACT-GAS mice without celecoxib treatment were also examined. The thickness of the gastric mucosa, cell populations, COX-2 expression, and PGE(2) levels were evaluated. All ACT-GAS mice showed gastric mucosal hypertrophy, and four of six 80-wk-old ACT-GAS mice developed gastric cancer. COX-2 was expressed in interstitial cells of the hypertrophic gastric mucosa and gastric cancers. Moreover, PGE(2) levels in the gastric mucosa of ACT-GAS mice were significantly higher than those of normal mice. With treatment with celecoxib, PGE(2) levels, the gastric mucosal thickness, and the number of total gastric cells per gastric gland of ACT-GAS mice were significantly decreased. The decrease in gastric mucosal thickness was caused by a reduction of foveolar hyperplasia. The thickness of glandules and the number of Ki67-positive cells were not significantly changed. In conclusion, COX-2 contributes to gastrin-induced mucosal hypertrophy of the stomach.

STAT3 is constitutively activated and supports cell survival in association with survivin expression in gastric cancer cells.

Signal transduction and activator of transcription 3(STAT3) signaling is constitutively activated in various tumors, and is involved in cell survival and proliferation during oncogenesis. There are few reports, however, on the role of STAT3 signaling in gastric cancer. The aim of the present study was to clarify the role of STAT3 signaling in apoptosis and cellular proliferation in gastric cancer. Here we reported that STAT3 was constitutively activated in various human gastric cancer cells and its inhibition by ectopic dominant-negative STAT3 or Janus kinase inhibitor, tyrphostin AG490, induced apoptosis. Furthermore, STAT3 inhibition markedly decreased survivin expression, and forced expression of survivin rescued AGS cells from apoptosis induced by STAT3 inhibition. Although some reports demonstrated that the PI3K/Akt pathway regulates survivin expression, inhibition of the PI3K/Akt pathway did not affect survivin expression in AGS and MKN1 cells. Finally, activated form of STAT3, Tyr-705 phospho-stat3, was found in the nucleus of cancer cells in 11 of 40 (27.5%) human gastric cancer specimens. These findings suggest that constitutively activated STAT3 signaling supports gastric cancer cell survival in association with survivin expression.2004

Differentiation-inducing factor-1 (DIF-1) inhibits STAT3 activity involved in gastric cancer cell proliferation via MEK-ERK-dependent pathway.

Differentiation-inducing factor-1 (DIF-1) is a chlorinated hexaphenone isolated from Dictyostelium. DIF-1 exhibits antitumor activity in several types of mammalian tumor cells, although the underlying mechanisms remain unknown. On the other hand, recent studies indicate that constitutively activated STAT3 acts as an oncogene and could be a target for antitumor drug. In the present study, we examined the effects of DIF-1 on proliferation of gastric cancer cell lines as well as on its signal transduction pathways, focusing mainly on STAT proteins. DIF-1 inhibited proliferation of gastric cancer cells. Western blot analysis and electrophoretic mobility shift assay showed that DIF-1 inhibited STAT3 activity in an MEK-ERK-dependent manner in gastric cancer cell lines, AGS and MKN28. Moreover, blockade of STAT3 activity by ectopic expression of dominant-negative STAT3 or the Janus kinase inhibitor, tyrphostin AG490, inhibited cell growth of AGS cells. These results suggest that STAT3 activity plays an important role for cell growth in AGS cells, and raises the possibility that inhibition of STAT3 activity is one of the mechanisms responsible for the antitumor effect of DIF-1 in these cells.

Prohormone convertase furin has a role in gastric cancer cell proliferation with parathyroid hormone-related peptide in a reciprocal manner.

We investigated the expression of parathyroid hormone-related peptide (PTHrP) and the relationship between PTHrP and its endoprotease furin in gastric cancer. PTHrP was colocalized with furin in 75% of gastric cancer tissues (six of eight) from patients with high serum PTHrP levels. PTHrP mRNA expression was confirmed in 67% of gastric cancer cell lines (four of six), whereas furin mRNA was detected in all six gastric cancer cell lines. In a cultured gastric cancer cell line, MKN28, mature PTHrP protein expression was markedly increased by transfection of furin cDNA. Furin cDNA-transfected MKN28 cells grew faster than did the mock controls. Moreover, furin mRNA expression in cultured gastric cancer cells was enhanced when PTHrP was added to the culture medium. These results suggest a link between PTHrP and furin in the regulation of gastric cancer cell growth. Furin might be involved not only in the production of the mature form of PTHrP, but also in promoting growth in gastric cancer cells.

Gastrin stimulates the growth of gastric pit cell precursors by inducing its own receptors.

Gastrin/CCK-B receptors (CCKB-Rs) are present on parietal and enterochromaffin-like cells in the gastric mucosa but not on pit cells in the proliferative zone. Because serum gastrin levels are well correlated with the growth of the gastric pit, we examined whether pit precursor cells express CCKB-Rs using hypergastrinemic transgenic mice and a mouse pit precursor cell line, GSM06. In situ hybridization indicated that CCKB-R mRNA was limited to the lower one-third of the mucosa in control mice, whereas it was faintly distributed along the mid- to low glandular region in the hypergastrinemic transgenic mouse mucosa. CCKB-R-positive midglandular cells appear to have a pit cell lineage; therefore, GSM06 cells were used for an [(125)I]gastrin binding study. [(125)I]gastrin bound to the membrane fraction of the GSM06 cells when precultured with gastrin. Gastrin dose dependently induced CCKB-R expression in GSM06 cells and stimulated their growth. Thus these findings suggest that gastrin directly stimulates the growth of the pit cell lineage by inducing its own receptor in pit cell precursors.