1.Suppression Of Hypoxia-inducible Factor-1α By Small Interfering RNA Effect The Glycolysis In Esophageal Carcinoma2.Safety And Tolerability Of Bismuthyl Ecabet For Suspension In Healthy Chinese Subjects

Backgroud:Esophageal carcinoma (EC) is one of the commonest cause of tumour-related deatharound the world. In China, ESCC (Esophageal squmaous cell carcinoma) morbidityand mortality ranks first in the world. The growth of solid tumors is rapid, its internalblood and oxygen supply is relatively insufficient. Hypoxia condition is a commonfeature of tumor environment. Tumor cells dependent on glycolysis for energy ischaracterized by its tolerance for hypoxia metabolic basis. Hypoxia-induciblefactor-1(HIF-1) is a transcription factor which present in most of mammal and humantissue in the hypoxia condition. As a key regulator to adapt tumor microcirculation,HIF-1α is over expressed in ESCC, significantly correlated with depth of tumorinvasion, tumor lymph node metastasis and organ metastasis. HIF-1α gene may playan important role in tumor glycolysis, and it has become the hot point of the researchabout tumorous aetiology and treatment.Objetives:To investigate the effect of RNA interference-based silencing of HIF-1α gene on theexpression of glycolysis associated genes in esophageal squamous carcinoma cellsTE13and Eca-109, and to explore the relation between HIF-1α and tumor glycolysis.Methods: 1. Esophageal squamous cancer TE13and Eca109cells were incubated undernormoxic and hypoxic conditions for different time (6h,12h,24h,48h). Expression ofHIF-1α protein was detected by Western blot, thus the most suitable hypoxicincubated time was selected.2. TE13, TE13/shRNA cells were incubated under normoxic and hypoxic conditionsfor12h. Then the expression of glycolysis associated genes(HK-Ⅱ、Glut-1、LDH-A)was measured on mRNA level by Realtime PCR.3. TE13, TE13/shRNA cells were incubated under normoxic and hypoxic conditionsfor12h. Then the expression of glycolysis associated genes(HK-Ⅱ、Glut-1、LDH-A)was measured on protein level by Western blot.4. Eca-109, Eca-109/shRNA cells were incubated under normoxic and hypoxicconditions for12h. Then the expression of glycolysis associated genes(HK-Ⅱ、Glut-1、LDH-A) was measured on mRNA level by Realtime PCR.5. Eca-109, Eca-109/shRNA cells were incubated under normoxic and hypoxicconditions for12h. Then the expression of glycolysis associated genes(HK-Ⅱ、Glut-1、LDH-A) was measured on protein level by Western blot.6. TE13, TE13/shRNA, Eca-109, Eca-109/shRNA cells were incubated undernormoxic and hypoxic conditions for12h. Then spectrophotometry were employed todetermin the content of lactic acid (LA) in the culture supernatant.Results:1. The expression of HIF-1α increasd after incubated in hypoxia environment,reaching its peak at12h, Thus,12h was selected as subsequent hypoxic incubationtime.2. The expression of HK-Ⅱ、Glut-1、LDH-A mRNA was significantly down-regulatedin HIF-1α silenced cells(P<0.05), and up-regulated in TE13、Eca-109cells underhypoxia, with significant differences(P<0.05). 3. The expression of HK-Ⅱ、Glut-1proteins was significantly down-regulated inHIF-1α silenced cells(P<0.05), and up-regulated in TE13、Eca-109cells underhypoxia, with significant differences(P<0.05). LDH-A protein expression was milddown-regulated in HIF-1α silenced cells(P＞0.05).4. TE13and Eca109secreted significantly more lactic acid when cells were cultured in hypoxiaenvironment. When HIF-1α was silenced, the lactic acid secretion decreased significantly (P＜0.05）．In HIF-1α silencing cells, hypoxia could not make the lactic acid secretion increase.Conclusions1. HIF-1α protein expression is related to exposure time under hypoxia condition,reaching its peak at12h.2. The expessions of HK-Ⅱ、Glut-1、LDH-A mRNA and protein were down-regulatedobviously when HIF-1α gene was silenced. HK-Ⅱ、Glut-1、LDH-A are maybe HIF-1αtargeting genes.3. Both in normoxia and hypoxia, silenceing HIF-1α gene can decrease the content oflactic acid in the culture supernatant. This passageway may relate to suppression of HK-Ⅱ、GLUT1and LDHA. Backgroud:Bismuthyl Ecabet (Patent Cooperation Treaty number: CN02/00387) issynthesized by compounding bismuth nitrate and sulfodehydroabietic acid. Thechemical structure is significantly different from that of Ecabet sodium. This novelagent is a combination of sulfodehydroabietic acid and bismuth, which forms a newkind of salt useful in in treating peptic ulcers and gastritis in animals. BismuthylEcabet could have a better effect in the treatment of peptic ulcers and gastritis thanthat of sulfodehydroabietic acid or salt of bismuth. In this study, we evaluated thesafety and tolerability of both single and repeated doses of Bismuthyl Ecabet forSuspension in healthy Chinese subjects to provide evidence to warrant further clinicaltrials.Objectives:The present study was designed and completed to assess the safety and tolerabilityof Bismuthyl Ecabet for Suspension in healthy Chinese subjects.Methods:For the study77volunteers were randomized into single-or multiple-dose groupsfor oral administration of200to1600mg of BE once or1200mg twice daily for7days.Safety and tolerability were assessed by adverse events, physical examinations andserum biochemistry, etc.Results:In both single-and multiple-dose studies, no severe adverse events were observedin any of the volunteers. The main adverse events caused by the drug in single dose groups were an increase in serum alanine transaminase (ALT),-glutamyltranspeptidase (GGT), blood urea nitrogen (BUN), a decrease in leukocytes anderythra. The percentages of adverse events judged to be possibly related to the drugwere2/18in400mg,3/18in800mg,2/8in1200mg, and none in the200,600or1600mg dose groups. In multiple-dose studies, a increased serum ALT and aspartictransaminase (AST) was found in one subject after7days of administration of thedrug. All the serum biochemistry and erythra returned to normal levels after7dayswithout special treatment.Conclusions:Bismuthyl Ecabet has proven to be safe and well tolerated in healthy Chinesesubjects. The oral dosing regime selected for subsequent Phase II/III clinical trialswas800mg, b.i.d., and a dose of up to1000mg, b.i.d.–may be proven to achieve abetter anti-ulcer effect.