MiR-1271 negatively regulates AKT/MTOR signaling and promotes apoptosis via targeting PDK1 in pancreatic cancer.

OBJECTIVE: Pancreatic cancer (PC) possesses a very poor prognosis, and its pathogenesis is not fully understood. Evidence has suggested that microRNAs play important roles in cancer development and progression, the present study was designed to study the function of miR-1271 in PC. PATIENTS AND METHODS: PC tissues and adjacent normal tissues were collected from 17 patients. MiR-1271 and PDK1 expression were quantified by quantitative reverse-transcriptional polymerase chain reaction (RT-PCR). AKT/MTOR signaling activity and PDK1 protein expression were determined by Western blot. Cell viability and apoptosis were assessed by MTT assay and enzyme-linked immunosorbent assay (ELISA). Luciferase assay was used to verify whether miR-1271 directly targets PDK1. RESULTS: MiR-1271 was significantly down-regulated in PC tissues compared with that in the paired normal adjacent tissue, and its expression was up-regulated dose-dependently upon cisplatin treatment in PC cells. Overexpression of miR-1271 in these cells produced a pro-apoptotic effect, similar to what caused by cisplatin treatment. Moreover, overexpression of miR-1271 inhibited AKT/MTOR signaling, which was due to the targeting relationship between miR-1271 and PDK1. Finally, knockdown of PDK1 exerted a similar effect on apoptosis to that of miR-1271 overexpression. CONCLUSIONS: MiR-1271 is a potent tumor suppressor in PC, its pro-apoptotic function was partially mediated by reduced AKT/MTOR signaling. Targeting miR-1271 may represent an effective strategy for PC treatment.

Eicosapentaenoic acid attenuates dexamethasome-induced apoptosis by inducing adaptive autophagy via GPR120 in murine bone marrow-derived mesenchymal stem cells.

Long-term use of glucocorticoids is a widespread clinical problem, which currently has no effective solution other than discontinuing the use. Eicosapentaenoic acid (EPA), an omega-3 long chain polyunsaturated fatty acid (n-3 PUFA), which is largely contained in fish or fish oil, has been reported to promote cell viability and improve bone metabolism. However, little is known about the effects of EPA on dexamethasome (Dex)-induced cell apoptosis. In this study, we showed that EPA-induced autophagy of murine bone marrow-derived mesenchymal stem cells (mBMMSCs). Meanwhile, EPA, but not arachidonic acid (AA), markedly inhibited Dex-induced apoptosis and promoted the viability of mBMMSCs. We also observed that EPA-induced autophagy was modulated by GPR120, but not GPR40. Further experiments showed that the mechanism of EPA-induced autophagy associated with GPR120 modulation involved an increase in the active form of AMP-activated protein kinase and a decrease in the activity of mammalian target of RAPA. The protective effect of EPA on Dex-induced apoptosis via GPR120-meditated induction of adaptive autophagy was supported by in vivo experiments. In summary, our findings may have important implications in developing future strategies to use EPA in the prevention and therapy of the side effects induced by long-term Dex-abuse.

Supracondylar extension fracture of the humerus in children. Manipulative reduction, immobilisation and fixation using a U-shaped plaster slab with the elbow in full extension.

We present a method of manipulative reduction, immobilisation and fixation using a U-shaped plaster with the elbow in extension for extension-type supracondylar fractures of the humerus in children. When the elbow is in full extension, both the extensor and the flexor muscles are neutralised during manipulative reduction and the carrying angle can be easily assessed thus preventing cubitus varus, the most common complication. In order to evaluate the efficiency of this method, we compared the clinical results of the new method with those of conventional treatment. In a group of 95 children who sustained an extension-type supracondylar fracture of the humerus, 49 were treated by the new method and 46 by the conventional method, reduction and immobilisation in a plaster slab with the elbow in flexion. Reduction and immobilisation were easily achieved and reliably maintained by one manipulation for all the children treated by the new method. In 12 children treated by the conventional method, the initial reduction failed and in seven secondary displacement of the distal fragment occurred during the period of immobilisation in plaster. All required a second or third manipulation. Of the 46 children, 28 (60.9%) had developed cubitus varus at a mean follow-up of 4.6 years when treated by the conventional method. None of the children treated by the new method developed cubitus varus. The mean score, according to the Hospital for Special Surgery (HSS) elbow scoring system, was 91 points using the new method and 78 with the conventional method. The results were statistically significant with regard to the incidence of cubitus varus and the elbow score (p < 0.01) suggesting that the new method is reliable and gives a satisfactory outcome.

The effect of endoscopic sphincterotomy on acute and chronic complications of biliary endoprostheses.

BACKGROUND: Endoscopically placed biliary stents have become routine therapy for bile duct obstruction and bile leaks. Controversy exists regarding the use of biliary sphincterotomy to facilitate placement of 10F plastic stents. METHODS: We retrospectively studied the effect of sphincterotomy on acute and chronic complications of 10F stent therapy. Data for acute complications, 30-day mortality and stent migration were obtained for 130 patients undergoing placement of a single 10F plastic biliary stent. For 109 patients in whom prolonged stent therapy was undertaken, the occurrence of and time to stent dysfunction were also analyzed. Sphincterotomy was performed in 48 cases (36.9%) based on physician preference. RESULTS: There were no failures in stent placement. The incidence of acute complications was higher in patients undergoing sphincterotomy (8.3% vs. 1.2%, p = 0.04). Stent migration was more common in the no sphincterotomy group versus the sphincterotomy group (8.5% vs. 0, p = 0.03). CONCLUSIONS: Sphincterotomy is not necessary for placement of 10F plastic stents and increases acute procedural morbidity. Interestingly, a higher incidence of stent migration was seen in patients who did not undergo biliary sphincterotomy.

Inhibition of caspase activity does not prevent the signaling phase of apoptosis in prostate cancer cells.

BACKGROUND: Caspases are a family of cysteine proteases capable of characteristically cleaving after an aspartic acid residue. Various members of the caspase family (e.g., caspases 8 and 9) have been implicated as critical initiators in the signaling phase, while others (e.g., caspases 3, 6, and 7) have been implicated in the effector or execution phase of apoptosis. Thapsigargin (TG) is capable of inducing cell proliferation-independent apoptosis of prostate cancer cells. This study was undertaken to determine if caspase inhibition can prevent TG- or 5-fluorodeoxyuridine (5-FrdU)-induced apoptosis in prostate cancer cells. METHODS: Caspase activity was evaluated by Western blot analysis of the cleavage of retinoblastoma (Rb) protein, a caspase substrate during TG-induced death of prostate cancer cells. In addition, hydrolysis of caspase-specific fluorescent peptide substrates was assayed in lysates from TG-treated cells. Clonogenic survival assays were performed following treatment of rat AT3 and human TSU-Pr1 prostate cancer cell lines with TG and 5-FrdU in the presence and absence of peptide caspase inhibitors. AT3.1 cells transfected with the crmA gene, encoding a viral protein with caspase-inhibitory activity, were also tested for clonogenic survival following TG and 5-FrdU exposure. RESULTS: During treatment with TG, Rb is first dephosphorylated and then proteolytically cleaved into 100-kDa and 40-kDa forms, indicative of caspase activity. A 6-8-fold increase in class II (i.e., caspases 3, 7, and 10) hydrolysis of the caspase substrate Z-DEVD-AFC was observed after 24 hr of TG or 5-FrdU. AT3 cells expressing crmA (i.e., an inhibitor of caspases 1, 4, and 8) were not protected from apoptosis induced by TG or 5-FrdU. The caspase inhibitors Z-DEVD-fmk (i.e., an inhibitor of caspases 3, 7, and 10) and Z-VAD-fmk (i.e., a general caspase inhibitor) were also unable to protect TSU and AT3 cells from apoptosis induced by TG or 5-FrdU. CONCLUSIONS: Caspase activation may play a role in the downstream effector phase of the apoptotic cascade; however, in this study, caspase inhibition did not prevent the signaling phase of apoptosis induced by two agents with distinct mechanisms of cytotoxicity, TG or 5-FrdU. These results suggest that caspase inhibition by recently described endogenous caspase inhibitors should not lead to development of resistance to TG. A strategy for targeting TG's unique cytotoxicity to metastatic prostate cancer cells is currently under development.

Mechanism and role of growth arrest in programmed (apoptotic) death of prostatic cancer cells induced by thapsigargin.

BACKGROUND: More than 95% of metastatic androgen independent prostatic cancer cells per day are in a proliferatively quiescent G0 state [Berges et al.: Clin Cancer Res 1:473-480, 1995] limiting their responsiveness to anti-proliferative chemotherapeutic agents. Novel therapeutics capable of activating the programmed (apoptotic) death pathway in these cells without requiring entrance into the proliferative cell cycle are urgently needed. Thapsigargin (TG) treatment of rapidly growing androgen independent prostatic cancer cells arrests such cells in G0 and induces their programmed death. This raises not only the issue of the mechanism for such growth arrest, but also whether this programmed death is simply a response of rapidly growing cells to growth arrest making cytotoxicity still dependent upon the initial rate of cell proliferation. METHODS: To resolve the mechanism of TG induced growth arrest, rat AT3.1 prostatic cancer cells were analyzed for RNA and protein expression of the growth arrest gene, gadd153, intracellular free Ca2+ levels (Cai), and cell cycle distribution on exposure to TG alone and in combination with Ca2+ chelation induced by BAPTA-AM or BAPTA-AM/EGTA. To resolve whether growth arrest is required for TG cytotoxicity, primary cultures of proliferatively quiescent, human prostatic cancer cells were exposed to TG. RESULTS: Co-treatment of androgen independent AT-3 rat prostatic cancer cells with the Cai chelator BAPTA plus TG prevented growth arrest, as monitored by DNA flow cytometry, and failure to induce mRNA and protein for gadd153, demonstrating that growth arrest is due to Cai elevation, not depletion of intracellular Ca2+ pools. In addition, proliferatively quiescent G0 primary cultures of human prostatic cancer cells were resistant to anti-proliferative agents, but could be induced to undergo programmed death by TG as documented by morphological criteria and 14C-labeled DNA fragmentation assays. CONCLUSIONS: These results demonstrate that TG with its ability to elevate Cai induces proliferating prostate cancer cells to growth arrest. Such Cai dependent growth arrest is not required, however, since TG can induce the programmed death of proliferatively quiescent G0 prostatic cancer cells without requiring either growth arrest or progression through the proliferative cell cycle.

Role of programmed (apoptotic) cell death during the progression and therapy for prostate cancer.

Cells possess within their epigenetic repertoire the ability to undergo an active process of cellular suicide termed programmed (or apoptotic) cell death. This programmed cell death process involves an epigenetic reprogramming of the cell that results in an energy-dependent cascade of biochemical and morphologic changes (also termed apoptosis) within the cell, resulting in its death and elimination. Although the final steps (i.e., DNA and cellular fragmentation) are common to cells undergoing programmed cell death, the activation of this death process is initiated either by sufficient injury to the cell induced by various exogenous damaging agents (e.g., radiation, chemicals, viruses) or by changes in the levels of a series of endogenous signals (e.g., hormones and growth/survival factors). Within the prostate, androgens are capable of both stimulating proliferation as well as inhibiting the rate of the glandular epithelial cell death. Androgen withdrawal triggers the programmed cell death pathway in both normal prostate glandular epithelia and androgen-dependent prostate cancer cells. Androgen-independent prostate cancer cells do not initiate the programmed cell death pathway upon androgen ablation; however, they do retain the cellular machinery necessary to activate the programmed cell death cascade when sufficiently damaged by exogenous agents. In the normal prostate epithelium, cell proliferation is balanced by a equal rate of programmed cell death, such that neither involution nor overgrowth normal occurs. In prostatic cancer, however, this balance is lost, such that there is greater proliferation than death producing continuous net growth. Thus, an imbalance in programmed cell death must occur during prostatic cancer progression. The goal of effective therapy for prostatic cancer, therefore, is to correct this imbalance. Unfortunately, this has not been achieved and metastatic prostatic cancer is still a lethal disease for which no curative therapy is currently available. In order to develop such effective therapy, an understanding of the programmed death pathway, and what controls it, is critical. Thus, a review of the present state of knowledge concerning programmed cell death of normal and malignant prostatic cells will be presented.

Androgen ablation-induced programmed death of prostatic glandular cells does not involve recruitment into a defective cell cycle or p53 induction.

Proliferating cells characteristically undergo programmed (i.e. apoptotic) death if their progression through the cell cycle is sufficiently perturbed. To determine whether androgen ablation-induced programmed death of prostatic glandular cells involves apoptosis triggered by recruitment of nonproliferating cells into a perturbed cell cycle, rat ventral prostates were assessed temporally after castration for several stereotypical molecular stigmata of entry into the proliferative cell cycle. Northern blot analysis was used to assess levels of transcripts from genes characteristically activated 1) during the transition from quiescence (G(0)) into G1 of the proliferative cell cycle (cyclin-D1 and cyclin-C), 2) during the transition from G1 to S (cyclin-E, cdk2, thymidine kinase, and H4-histone), and 3) during progression through S (cyclin-A). Although levels of each of these transcripts increased as expected in prostatic glandular epithelial cells stimulated to proliferate by the administration of exogenous androgen to previously castrated rats, levels of the same transcripts decreased in prostatic glandular cells induced to undergo apoptosis after androgen withdrawal. Northern and Western blot analyses also demonstrated that there was no increase in prostatic p53 messenger RNA or protein content per cell after androgen ablation. Likewise, after castration, there was no enhanced prostatic expression of the WAF1/CIP1 gene, a gene whose expression is known to be induced in both a p53-dependent and -independent manner during recruitment from G0 into G1. In addition, androgen ablation-induced apoptosis of prostatic glandular cells was not accompanied by retinoblastoma protein phosphorylation, which is characteristic of progression into late G1. Nuclear run-on assays demonstrated that there was no increase in the prostatic rate of transcription of the c-myc and c-fos genes after castration. These results demonstrate that prostatic glandular cells undergo programmed death in G(0) without recruitment into the G1 phase of a defective cell cycle, and that an increase in p53 protein or its function is not involved in this death process.

[The expression and significance of blood group antigens (BGA) A, B, H, Le(a) and Le(b) in hepatocellular carcinoma and chronic hepatitis].

Five monoclonal antibodies which selectively recognize A, B, H, Le(a) and Le(b) BGA had been used for immunohistochemical examination of serial sections collected from 40 cases of hepatocellular carcinomas, 63 cases of chronic hepatitis, 10 cases each of fetal and normal adult liver. All of the cases had been followed-up except the autopsy cases. The results were as follows: in fetal and normal adult liver tissues, none of the liver BGA were detected in the parenchymal liver cells. Five BGA were expressed in 11 hepatitic cases (17%) and also expressed in 19 hepatocellular carcinomas (48%). Combining the expression of BGA with the follow-up results, the study we have performed indicates that the expression of BGA in chronic hepatitis predicts cancerous change or severe liver cell damage associated with poor prognosis.

Protease II from Escherichia coli: sequencing and expression of the enzyme gene and characterization of the expressed enzyme.

Protease II gene of Escherichia coli HB101 was cloned and expressed in E. coli JM83. The transformant harboring a hybrid plasmid, pPROII-12, with a 2.4 kbp fragment showed 90-fold higher enzyme activity than the host. The whole nucleotide sequence of the inserted fragment of plasmid pPROII-12 was clarified by the dideoxy chain-terminating method. The sequence that encoded the mature enzyme protein was found to start at an ATG codon, as judged by comparison with amino terminal protein sequencing. The molecular weight of the enzyme was estimated to be 81,858 from the nucleotide sequence. The reactive serine residue of protease II was identified as Ser-532 with tritium DFP. The sequence around the serine residue is coincident with the common sequence of Gly-X-Ser-X-Gly, which has been found in the active site of serine proteases. Except for this region, protease II showed no significant sequence homology with E. coli serine proteases, protease IV and protease La (lon gene), or other known families of serine proteases. However, 25.3% homology was observed between protease II and prolyl endopeptidase from porcine brain. Although the substrate specificities of these two enzymes are quite different, it seems possible to classify protease II as a member of the prolyl endopeptidase family from the structural point of view.

Cloning and sequencing of the 7 alpha-hydroxysteroid dehydrogenase gene from Escherichia coli HB101 and characterization of the expressed enzyme.

The 7 alpha-hydroxysteroid dehydrogenase (EC 1.1.1.159) gene from Escherichia coli HB101 was cloned and expressed in E. coli DH1. The hybrid plasmid pSD1, with a 2.8-kbp insert of chromosomal DNA at the BamHI site of pBR322, was subcloned into pUC19 to construct plasmid pSD3. The entire nucleotide sequence of an inserted PstI-BamHI fragment of plasmid pSD3 was determined by the dideoxy chain-termination method. Within this sequence, the mature enzyme protein-encoding sequence was found to start at a GTG initiation codon and to comprise 765 bp, as judged by comparison with the protein sequence. The deduced amino acid sequence of the enzyme indicated that the molecular weight is 26,778. The transformant of E. coli DH1 harboring pSD3 with a 1.8-kbp fragment showed about 200-fold-higher enzyme activity than the host. The enzyme was purified by a single chromatography step on DEAE-Toyopearl and obtained as crystals, with an activity yield of 39%. The purified enzyme was homogeneous, as judged by sodium dodecyl sulfate gel electrophoresis. The enzyme was most active at pH 8.5 and stable between pH 8 and 9. The enzyme was NAD+ dependent and had a pI of 4.3. The molecular mass was estimated to be 120 kDa by the gel filtration method and 28 kDa by electrophoresis, indicating that the enzyme exists in a tetrameric form.

Prognostic significance of general immune competence in acute leukemia.

The prognostic significance of lymphocyte transformation rate, E-RFC%, 29 degrees C E-RFC%, serum IgG, IgA, IgM and C3 levels were studied in 286 patients with acute leukemia. In acute lymphoblastic leukemia (ALL), the pretreatment immunological parameters were not related to whether the patients could achieve complete remission (CR), but the patients with E-RFC% greater than 30% before treatment or E-RFC% restored to normal after treatment had a significantly longer survival time. In acute nonlymphocytic leukemia (ANLL), pretreatment LTR and E-RFC% were significantly higher in patients who could achieve CR subsequently, the patients with higher pretreatment levels of LTR, E-RFC% or 29 degrees C E-RFC% survived significantly longer than patients with lower levels of these parameters, and the patients whose LTR could return to normal after treatment had a significantly higher CR rate and longer survival times. Except that the pretreatment IgM level was related to the survival of ANLL patients, serum levels of immunoglobulins and C3 had no prognostic value for both ALL and ANLL. In both ALL and ANLL, the immunological parameters changed significantly when relapse occurred.

Extraovarian pelvic serous tumor with marked calcification.

We report two cases of low-grade extraovarian pelvic serous tumor. Each contained large numbers of psammoma bodies. The tumors belong to the small group of serous carcinomas that arise from the peritoneum. In both cases the tumor permeated the myometrial lymphatic vessels extensively. One patient is well eight years after surgery.

Significance of residual tumor at the esophageal stump after resection for carcinoma.

Residual tumor at the esophageal stump was found in 103, or 14.3%, of a series of 719 resected esophageal specimens for carcinoma. The residual tumor pathologically presented as basal epithelial-cell carcinomatous change in 16 (15.5%), carcinoma in situ in 26 (25.5%), invasive carcinoma in 51 (49.5%), and cancerous emboli in 6 (5.8%) cases. A follow-up study of the 719 patients showed that the long-term survival rate of those with residual tumor was significantly lower than that of those without. Factors of occurrence besides inadequate resection were analyzed, and indications of surgical treatment were recommended. The presence of residual tumor is not an important factor in the development of anastomotic leakage.