ABSTRACT
BACKGROUND: This study was designed to explore the anticancer potential of isoalantolactone, a sesquiterpene lactone, on esophageal squamous cell carcinoma (ESCC) cells and associated molecular mechanisms. METHODS: ESCC cell lines were treated with isoalantolactone or vehicle and tested for viability, proliferation, cell cycle distribution, and apoptosis. Xenograft tumor studies in nude mice were done to examine the in vivo anticancer effect of isoalantolactone. RESULTS: Isoalantolactone treatment reduced ESCC cell viability and proliferation in vitro, which was coupled with induction of G0/G1 cell cycle arrest and apoptosis. In vivo studies confirmed the growth-suppressive effect of isoalantolactone on ESCC cells. Mechanistically, isoalantolactone reversed microRNA-21-mediated repression of programmed cell death 4 (PDCD4). Overexpression of microRNA-21 and knockdown of PDCD4 blocked the growth suppression and apoptosis induction by isoalantolactone in ESCC cells. CONCLUSIONS: Isoalantolactone shows growth-suppressive activity against ESCC cells, which is ascribed to upregulation of PDCD4 via downregulation of microRNA-21.
Subject(s)
Antineoplastic Agents/pharmacology , Apoptosis Regulatory Proteins/metabolism , Carcinoma, Squamous Cell/drug therapy , Cell Proliferation/drug effects , Esophageal Neoplasms/drug therapy , MicroRNAs/metabolism , RNA-Binding Proteins/metabolism , Sesquiterpenes/pharmacology , Animals , Apoptosis/drug effects , Apoptosis Regulatory Proteins/genetics , Carcinoma, Squamous Cell/enzymology , Carcinoma, Squamous Cell/genetics , Carcinoma, Squamous Cell/pathology , Cell Cycle Checkpoints/drug effects , Cell Line, Tumor , Dose-Response Relationship, Drug , Down-Regulation , Esophageal Neoplasms/enzymology , Esophageal Neoplasms/genetics , Esophageal Neoplasms/pathology , Esophageal Squamous Cell Carcinoma , Gene Expression Regulation, Neoplastic , Humans , Male , Mice, Inbred BALB C , Mice, Nude , MicroRNAs/genetics , RNA-Binding Proteins/genetics , Signal Transduction/drug effects , Time Factors , Tumor Burden/drug effects , Xenograft Model Antitumor AssaysABSTRACT
BACKGROUND AND AIM: Manometric studies on the human lower esophageal sphincter (LES) have shown radial asymmetry of the high-pressure zone (HPZ). The aim of this study was to compare the functional properties of human LES clasp and sling muscles, and to look at their relationship with the expression of muscarinic receptors and intracellular Ca(2+) concentration. METHODS: Muscle strips of sling and clasp fibers from the LES were obtained from patients undergoing subtotal esophagectomy. Isometric tension responses of the strips to acetylcholine were studied. Western blotting and reverse transcription-polymerase chain reaction (RT-PCR) were used to determine the expression of five subtypes of muscarinic receptors. Intracellular Ca(2+) ([Ca(2+) ]i) was measured using laser scanning confocal microscopy. RESULTS: Acetylcholine caused a concentration-dependent increase in the tension of sling and clasp strips, the sling strip being stronger than clasp (P=0.00). Messenger RNA and protein for the five muscarinic acetylcholine receptor (mAChRs) expressed in the sling and clasp muscles were highest for M2, and then in decreasing levels: M(3)>M(1)>M(4)>M(5) . Acetylcholine caused significant elevation of [Ca(2+) ]i in sling and clasp muscle cells in the presence of extracellular Ca2+ (1.5mmol/L), and Ach-induced [Ca(2+) ]i elevation was 1.6 times greater in sling cells than in clasp cells. CONCLUSION: Variation of intracellular concentrations of Ca(2+) may be the reason for differential responses to acetylcholine for sling versus clasp fibers. However, these differences are not associated with the distribution and the level of expression of the five mAChRs between the two muscle types. Further study should focus on the ligand affinity and signal transduction pathway.
Subject(s)
Acetylcholine/pharmacology , Cholinergic Agonists/pharmacology , Esophageal Sphincter, Lower/drug effects , Muscle Contraction/drug effects , Receptors, Muscarinic/drug effects , Aged , Blotting, Western , Calcium/metabolism , Calcium Channel Blockers/pharmacology , Dose-Response Relationship, Drug , Esophageal Sphincter, Lower/innervation , Esophageal Sphincter, Lower/metabolism , Female , Humans , In Vitro Techniques , Male , Microscopy, Confocal , Middle Aged , Nifedipine/pharmacology , RNA, Messenger/metabolism , Receptors, Muscarinic/genetics , Receptors, Muscarinic/metabolism , Reverse Transcriptase Polymerase Chain Reaction , Time FactorsABSTRACT
BACKGROUND AND AIM: Cholecystokinin (CCK) and gastrin exert their influences via CCK receptors. This research was conducted to look at the responses of the sling and clasp fibers forming the human lower esophageal sphincter (LES) to CCK and gastrin, and the role of CCK receptors in the responses. METHODS: Muscle strips of sling and clasp fibers from the LES were obtained from patients undergoing subtotal esophagectomy. Isometric tension responses of the strips to CCK-8 and gastrin-17 were studied, and the maximum effect (E(max)) for each agonist was derived. CCK-A receptor antagonist, CR1409 and CCK-B antagonist, CR2945 were applied to sling and clasp fibers and their pK(B) values were calculated. RESULTS: Sling fibers produced significant contractions following exposure to CCK-8 and gastrin-17, while clasp fibers had less responses to the two agents. CR1409 and CR2945 inhibited responses of sling to CCK-8 in a concentration-dependent fashion. The inhibition effects of the two antagonists on clasp fibers were not measurable because there was a mild contraction of the fiber in response to CCK-8. CONCLUSION: The contractions generated by sling fibers following exposure to CCK and gastrin are greater than that produced by clasp fibers. CCK-A receptors are more important for the generation of contractions by the sling fibers, whereas both CCK-A and CCK-B receptors are involved in the functional regulation of the clasp fibers. [Corrections added after online publication 28 April 2008: in the Background and Aims section of the preceding abstract, all instances of 'CKK' were corrected to 'CCK'. In the final sentence of the abstract 'CCKA'was corrected to 'CCK-A'. In the article title '(CKK)' was corrected to '(CCK)'.].
