Effect of verapamil in the reversal of doxorubicin chemotherapy resistance in advanced gastric cancer.

OBJECTIVE: Gastric carcinoma is one of the most common malignant tumors and one of the most common cancer-related fatal diseases. Chemotherapy is considered as the major therapy for advanced gastric cancer, and the curative effect of chemotherapy directly affects the treatment of advanced gastric cancer. Drug resistance of tumor cells is one of the important causes that induces failure of chemotherapy. Previous studies have demonstrated that verapamil (VER) can reverse drug resistance by inhibiting the P-glycoprotein (P-gp), which is one of the main targets of VER. The present study aimed at investigating the function of glucosylceramide synthase (GCS) in the VER-induced reversal of doxorubicin (ADM) chemotherapy resistance in gastric carcinoma. PATIENTS AND METHODS: In the current study, the 4 GC cell line was selected for investigation. The IC50 values of gastric cancer cells were measured using CCK-8 assay. The expression levels of candidate genes in gastric carcinoma cells were measured by RT-qPCR. The expression levels of candidate protein in gastric carcinoma cells were measured by Western blot. The expression of GCS protein in clinical specimens of GC receiving VER+TACE therapy was measured by immunohistochemistry. The test of gastric carcinoma cell apoptosis was measured by Annexin V-FITC/PI double-staining. RESULTS: We found that the expression levels changes of the GCS gene can influence the effects of ADM+VER on cell apoptosis. The role and mechanism of GCS gene in reversing the chemotherapy resistance of gastric carcinoma cells to ADM were explored. CONCLUSIONS: In future research, we will explore the mechanism of how GCS affects drug resistance in gastric carcinoma and related signal transduction pathway.

The clinical observation of verapamil in combination with interventional chemotherapy in advanced gastric cancer.

OBJECTIVE: We analyzed the clinical observations of target arterial infusion of verapamil combined with chemotherapy as therapy for advanced gastric cancer. PATIENTS AND METHODS: From March 2012 to December 2015, a total of 63 patients with advanced gastric cancer were admitted to our department. The target artery in the control group was perfused with chemotherapy drugs only, and the target artery in the therapy group was injected with verapamil combined with chemotherapy drugs. RESULTS: The therapeutic effect of the therapy group was significantly better than that of the control group in the primary foci of gastric cancer. Liver metastatic lesions: 11 patients in the control group had liver metastases and 25 patients in the therapy group had liver metastases. The effective rate (CR+PR) of the therapy group was significantly better than the control group. Clinical benefit evaluation: in the therapy group of 43 cases, 40 cases presented positive clinical benefit and 38 cases positive clinical weight in KFS scoring system; the clinical benefit of the therapy group was significantly better than control group. Survival analysis: the disease progression-free rate and survival rate of the therapy group were 12 months and 24 months, which were higher than those in the control group. The median PFS and median OS were also significantly longer than those in the control group (p<0.01). In the therapy group, adverse effects of chemotherapy in 43 patients were relieved in a short time. CONCLUSIONS: Target arterial infusion of verapamil combined with chemotherapy drugs for advanced gastric cancer can significantly improve the efficacy of chemotherapy drugs and prolong the survival of patients.

Down-regulation of miR-329-3p is associated with worse prognosis in patients with cervical cancer.

OBJECTIVE: miR-329-3p has been reported to serve as a tumor suppressor in the progression of cervical cancer (CC). The aim of this study was to investigate the clinical significance of miR-329-3p in human CC. PATIENTS AND METHODS: Quantitative RT-PCR (qRT-PCR) was used to detect miR-329-3p expression in CC tissue samples and matched normal cervical tissues. The x2 test was used to analyze the association between miR-329-3p expression and clinical features of CC patients. Moreover, we evaluated the prognostic value of miR-329-3p by Kaplan-Meier survival curve and Cox regression model. RESULTS: We found that the mean expression level of miR-329-3p in CC tissues was significantly lower than the mean level in the adjacent normal tissues samples (p < 0.01). MiR-329-3p level was significantly associated with lymph node metastasis (p = 0.013), FIGO stage (p = 0.024) and distant metastasis (p = 0.001). Furthermore, a significant difference was found, that CC patients with low miR-329-3p expression level had distinctly shorter overall survival than patients with high miR-329-3p expression level (p = 0.001). Finally, multivariate analyses indicated that miR-329-3p represented an independent predictor for overall survival of CC (p = 0.04). CONCLUSIONS: These results indicated, for the first time, that down-regulation of miR-329-3p was associated with poor prognosis in CC patients. MiR-329-3p can be used as an independent factor to predict survival of patients with CC.

Expression, purification, and biochemical characterization of the amino-terminal extracellular domain of the human calcium receptor.

We purified the extracellular domain (ECD) of the human calcium receptor (hCaR) from the medium of HEK-293 cells stably transfected with a hCaR cDNA containing an isoleucine 599 nonsense mutation. A combination of lectin, anion exchange, and gel permeation chromatography yielded milligram quantities of >95% pure protein from 15 liters of starting culture medium. The purified ECD ran as an approximately 78-kDa protein on SDS-polyacrylamide gel electrophoresis and was found to be a disulfide-linked dimer. Its NH2-terminal sequence, carbohydrate content, and CD spectrum were defined. Tryptic proteolysis studies showed two major sites accessible to cleavage. These studies provide new insights into the structure of the hCaR ECD. Availability of purified ECD protein should permit further structural studies to help define the mechanism of Ca2+ activation of this G protein-coupled receptor.

Mutational analysis of the cysteines in the extracellular domain of the human Ca2+ receptor: effects on cell surface expression, dimerization and signal transduction.

Mammalian calcium receptors (CaRs) share with the metabotropic glutamate receptors (mGluRs) the relative positions of 16 cysteine residues in the amino-terminal extracellular domain. To investigate the role of these cysteines, a series of mutants in the extracellular domain of the human CaR was prepared in which each of these 16 cysteine residues and three others not conserved in the mGluRs were replaced by serines. Wild-type and mutant CaR cDNAs were expressed in HEK-293 cells, and evaluated for expression and response to extracellular calcium. Mutation of three non-conserved cysteines and of two conserved cysteines produced proteins with near wild-type phenotype. In contrast, mutation of the other conserved cysteines gave proteins that showed drastic reduction in cell surface expression and/or failed to respond to calcium. We identified 14 cysteines essential for proper trafficking and function of the receptor, two of which may be involved in formation of a disulfide-linked dimer.

A Ca(2+)-sensing receptor mutation causes hypoparathyroidism by increasing receptor sensitivity to Ca2+ and maximal signal transduction.

Activating mutations of the Ca(2+)-sensing receptor (CaR) gene cause autosomal dominant hypoparathyroidism. Functional expression studies have been reported for several mutations, but have produced conflicting results. Thus, the mechanism by which these mutations activate the receptor is unclear. We describe here a new family with autosomal dominant hypoparathyroidism. The mother and three daughters experienced muscle spasms and/or seizures from early childhood. They were treated with oral calcium and vitamin D analogs, and all four patients developed hypercalciuria, nephrocalcinosis, and renal insufficiency. In this family, we identified a heterozygous missense mutation (F612S) involving the extracellular region of the CaR. The mutation cosegregated with disease. It was not present in 50 normal control individuals. We used site-directed mutagenesis to introduce this mutation into the CaR cDNA, and then expressed the mutant receptor in human embryonic kidney (HEK)-293 cells. In these cells, the accumulation of inositol phosphates was measured as a function of extracellular Ca2+ concentration. Compared with the wild-type receptor, the mutant receptor showed a left-shift in the concentration-response curve and an increase in the maximal response to high Ca2+ concentration. These effects did not appear to be mediated by changes in levels of receptor expression, as judged by ELISA, or by changes in receptor glycosylation, as judged by Western analysis. We conclude that this CaR mutation causes hypoparathyroidism by a dual increase in receptor sensitivity to extracellular Ca2+ and maximal signal transduction capacity.

Sporadic hypoparathyroidism caused by de Novo gain-of-function mutations of the Ca(2+)-sensing receptor.

Activating mutations of the Ca(2+)-sensing receptor (CaR) gene have been identified in families with autosomal dominant hypoparathyroidism and in one patient with sporadic hypoparathyroidism. Here, we describe two additional patients with sporadic hypoparathyroidism. One patient presented with mild symptoms at age 18 yr; the other was severely symptomatic from infancy. A heterozygous missense mutation was identified in each patient. One mutation (L773R) involved the fifth transmembrane domain of the CaR, the other (N118K) affected the amino-terminal, extracellular domain. In both cases, the probands' parents lacked the mutation, indicating that the mutations arose de novo. In expression studies the mutations shifted the concentration-response curve to the left and increased maximal activity. We conclude that 1) sporadic hypoparathyroidism can be caused by de novo gain-of-function mutations of the CaR; 2) the phenotype can vary from mild to life-threatening hypocalcemia; 3) gain-of-function mutations can involve not only extracellular regions, as previously reported, but also transmembrane domains of the CaR; and 4) the mechanism of activation can involve both increased receptor sensitivity to Ca2+ and increased maximal signal transduction.

The carboxyl terminus of the human calcium receptor. Requirements for cell-surface expression and signal transduction.

The G-protein-coupled calcium receptor plays a key role in extracellular calcium homeostasis. To examine the role of the membrane-spanning domains and the approximately 200-residue cytoplasmic carboxyl terminus of the calcium receptor in cell-surface expression and signal transduction, we transfected HEK-293 cells with a series of truncation and carboxyl-terminal missense mutants and analyzed expression by immunoblotting, glycosidase digestion, intact cell immunoassay, and extracellular calcium-stimulated phosphoinositide hydrolysis assay. Two truncation mutants terminating at residues 706 and 802 within the second and third intracellular loops, respectively, were not properly glycosylated, failed to reach the cell-surface, and showed no calcium response, indicating that mutant receptors with the full extracellular domain but only three or five transmembrane domains are improperly folded and/or processed. Truncation mutants terminating at residues 888 and 903 within the carboxyl terminus were equivalent to the wild type in all assays, whereas mutants truncated at residues 865 and 874 showed no response to calcium, despite only approximately 25% reduction in cell-surface expression. Mutants with a full-length carboxyl terminus but with residues between positions 874 and 888 replaced with alanines showed either no (Ala875, Ala876, and Ala879) or significantly reduced (Ala881-Ala883) calcium response at levels of cell-surface expression equivalent to those of the wild-type receptor. These results indicate that deletion of the majority of the carboxyl terminus is compatible with normal processing, cell-surface expression, and signal transduction of the receptor. The truncation and alanine substitution mutants identify a small region between residues 874 and 888 critical for normal signal transduction by the receptor.

Posterior bow and vanishing line signs in diagnosis of burst fractures of the spine on plain radiographs.

It is not uncommon to misdiagnose a burst fracture as a wedge compression fracture initially on plain film, resulting in a late progressive deformity and neurologic damage. The purpose of this study was to analyze the sensitivity, specificity and positive predictive value of plain radiographs in the diagnosis of thoracic and lumbar burst fractures using the posterior bow (PB) and vanishing line (VL) signs. Seven independent examiners, comprising three chief orthopedic residents, two radiologic third-year residents and two emergency attending physicians (orthopedists), randomly reviewed 26 sets of admission anteroposterior and lateral thoracolumbar spine radiographs taken in association with back injuries. They were asked to decide whether patients had a burst or a wedge compression fracture. All patients had computed tomography (CT) scans for diagnostic confirmation. The overall initial sensitivity using discriminant analysis in the diagnosis of burst fractures was 80%. This increased to 90% after the examiners were requested to use the PB and VL signs. The specificity decreased slightly from 75% to 71%, while the positive predictive value remained at 88%. Overdiagnosis of wedge compression fractures as burst fractures occurred, especially when the quality of the films was not ideal. We conclude that, with careful reading, the PB and VL signs help in identifying burst fractures on the initial plain film evaluation.

Effect of transmembrane Ca2+ gradient on the coupling of beta-adrenergic receptors and adenylyl cyclase.

In order to investigate the effect of transmembrane Ca2+ gradient on Gs mediated coupling of beta-AR and adenylyl cyclase, beta-AR from duck erythrocytes and Gs and adenylyl cyclase from bovine brain cortices were co-reconstituted into asolectin liposomes with different transmembrane Ca2+ gradient. These proteoliposomes were proven to be impermeable to water-soluble substances. The results obtained indicate that a physiological transmembrane Ca2+ gradient (1000-fold) is essential for higher stimulation of adenylyl cyclase by hormone-activated beta-AR via coupling to Gs and can be further enhanced by the decrease of such Ca2+ gradient within certain range (100 fold) following Ca2+ influx into cells during signal transduction. Fluorescence polarization of DPH revealed that transmembrane Ca2+ gradient modulates adenylyl cyclase and its stimulation by hormones through mediating a change in lipid fluidity. Correspondent conformational changes of beta-AR were also detected from the fluorescence spectra and quenching of Acrylodan-labelled beta-AR in those proteoliposomes. It is suggested that a proper transmembrane Ca2+ gradient is essential for the optimal fluidity of the phospholipid bilayer in the proteoliposomes, which favors the formation of a suitable conformation of the reconstituted beta-AR and thus promotes the stimulation of adenylyl cyclase activities by hormone-activated beta-AR via Gs.

Two-level burst fractures: clinical evaluation and treatment options.

Two-level burst fractures are rare. In a series of 180 surgically treated spinal fracture-dislocations, seven had such injuries, with an incidence of 3.9%. Four had fracture sites without contiguity: C4-T12 (one), L1-L4 (one), L2-L4 (one), and L2-L5 (one); and three with contiguity: T12-L1 (one), L1-L2 (one), and L2-L3 (one). L2 was the most frequently involved site, accounting for four in seven. Falling from height was the most common mechanism of injury, accounting for four in seven. Five in seven patients (71%) sustained multiple injuries. Chest traumas and extremity fractures were the ones most frequently associated. All of these patients had incomplete neurologic deficits at initial presentation. In the four discontiguous bursts, the neurologic levels corresponded to the cephalic ones. Six patients had follow-up periods of more than 2 years. Transpediculate systems were used in five, and at follow-up, two had screw breakages. In this series, the average neurologic recovery was 1.3 grades on the Frankel scale. In conclusion, it is mandatory to have a thorough organ system review when such patients are first seen. Then each fracture site would be judged seperately as either a stable or unstable burst preoperatively. Every effort should be made to treat any unstable segment via anterior, posterior, or combined approaches.

A follow-up study of porous-coated anatomic knee arthroplasty.

Ninety-four patients (105 knees) having a porous-coated anatomic total knee arthroplasty were retrospectively studied. The mean follow-up period was 5.8 years. The diagnoses were osteoarthritis (90.5%) and rheumatoid arthritis (9.5%). There were 80 women and 14 men. The mean age at operation was 58.5 years. Clinical evaluation, using the Hospital for Special Surgery knee score along with radiography, was used to assess knee status before and after surgery. The postoperative mean axial alignment was 3 degrees varus compared with 11 degrees varus before surgery. Placement of the prosthetic components was acceptable, with the femoral component in 6.5 degrees of valgus and the tibial component in 1.9 degrees of varus and 1.1 degrees posterior inclination. Complications included eight aseptic loosenings, four patellar maltrackings, one patellar fracture, eight wound problems, and one extension contracture. Most of the patients' knee function improved after surgery. The initial, postoperative range of motion improved in patients receiving continuous passive motion (CPM) immediately after surgery (P = .03) compared to patients without CPM application; however, the long-term follow-up data show no significance (P = .06) whether CPM was used or not. Age, body weight, degree of arthritic change, and modes of fixation yielded no significant influence on the final outcome. The porous-coated anatomic total knee arthroplasty is a valuable alternative procedure in the advanced arthritic knee when the proper candidates are selected, accurate surgical technique is executed and a suitable fixation mode is chosen.

Effect of transmembrane Ca2+ gradient on Gs function.

Gs and adenylate cyclase from bovine brain cortices were co-reconstituted into asolectin liposomes with or without 1000-fold transmembrane Ca2+ gradient. Obtained results showed that Gs activities of both binding GTP gamma S and stimulating adenylate cyclase were the highest in proteoliposomes, with a transmembrane Ca2+ gradient similar to the physiological situation and the lowest while the transmembrane Ca2+ gradient was in the inverse direction. Such a difference could be diminished following the dissipation of the transmembrane Ca2+ gradient by A23187. Time-resolved fluorescence anisotropy of diphenylhexatriene (DPH) has been used to compare the physical state of phospholipids among those proteoliposomes. It is suggested that a proper transmembrane Ca2+ gradient is essential for higher membrane fluidity, which may favor Gs function with higher GTP-binding activity and stimulation of adenylate cyclase.

Olecranon fractures treated with tension band wiring techniques--comparisons among three different configurations.

Retrospective comparisons were conducted among 3 different configurations of tension band wiring techniques for adult olecranon fractures. The follow-up period was at least one year (average 26 months). All 3 configurations achieved high union rates with an average of 3.4 months to achieve union. However, the olecranon screw group or Kirschner wire group had a relatively higher satisfactory rate than the Rush pin group, even though there was no significance in statistics (P > 0.05). Complications were few, and implant migration occurred in the Rush pin or Kirschner wire group. The complications were not difficult to manage. The authors conclude that for olecranon fractures with a large fragment, olecranon screw or Kirschner wire can be chosen. For olecranon fractures with a small fragment, Kirschner wire should be utilized.