In vitro modulation of Bcl-2 levels in small cell lung cancer cells: effects on cell viability

Small cell lung cancer (SCLC) is an aggressive disease, representing 15 percent of all cases of lung cancer, has high metastatic potential and low prognosis that urgently demands the development of novel therapeutic approaches. One of the proposed approaches has been the down-regulation of BCL2, with poorly clarified and controversial therapeutic value regarding SCLC. The use of anti-BCL2 small interfering RNA (siRNA) in SCLC has never been reported. The aim of the present study was to select and test the in vitro efficacy of anti-BCL2 siRNA sequences against the protein and mRNA levels of SCLC cells, and their effects on cytotoxicity and chemosensitization. Two anti-BCL2 siRNAs and the anti-BCL2 G3139 oligodeoxynucleotide (ODN) were evaluated in SCLC cells by the simultaneous determination of Bcl-2 and viability using a flow cytometry method recently developed by us in addition to Western blot, real-time reverse-transcription PCR, and cell growth after single and combined treatment with cisplatin. In contrast to previous reports about the use of ODN, a heterogeneous and up to 80 percent sequence-specific Bcl-2 protein knockdown was observed in the SW2, H2171 and H69 SCLC cell lines, although without significant sequence-specific reduction of cell viability, cell growth, or sensitization to cisplatin. Our results question previous data generated with antisense ODN and supporting the present concept of the therapeutic interest in BCL2 silencing per se in SCLC, and support the growing notion of the necessity of a multitargeting molecular approach for the treatment of cancer.

Relationship between Cx40/43 regulating the intracellular Ca2+ concentration of vascular smooth muscle cell (VSMC) and endothelium-dependent vascular contractive response of superior mesenteric artery in rats / 中华烧伤杂志

<p><b>OBJECTIVE</b>To investigate the relationship between intracellular Ca2+ concentration ([Ca2+]i) mediated by connexin 40/43( Cx40/43) of VSMC and endothelium-dependent vascular contractive response of superior mesenteric arteries (SMA) in hemorrhagic shock rats.</p><p><b>METHODS</b>Third to fifth passage culture of vascular endothelial cells (VEC) and VSMC from SD rats were used as study subject, the changes in contractive response of SMA and VSMC against hypoxia were observed. The expression of Cx40/43 in SMA,VEC,VSMC were blocked by Cx40/43 ASODN, then the effect of Cx40/43 on contractive response of hypoxic SMA and [Ca2+]i of VSMC were observed.</p><p><b>RESULTS</b>The contractive responses of SMA and VSMC after hypoxia were first increased, then decreased. Hypoxia induced calcium overload in VSMC [(82 +/- 4)% in normal control group, (115 +/- 8)% in hypoxia group at 30 min, (133 +/- 13)% in hypoxia group at 2 h]. Cx40 ASODN increased [Ca2+]i in VSMC and contractive response of SMA towards myricetin, while that of Cx43 ASODN showed opposite tendency.</p><p><b>CONCLUSIONS</b>Cx40/43 can regulate the SMA endothelium-dependent vascular contractive response through [Ca2+]i of VSMC after hemorrhagic shock.</p>

The mechanism of apoptosis in human U87 glioma cells induced by miR-21 antisense oligonucleotide / 中华医学遗传学杂志

<p><b>OBJECTIVE</b>To investigate the mechanism of U87 cell apoptosis induced by inhibiting miR-21 expression.</p><p><b>METHODS</b>Antisense oligonucleotides of miR-21 were chemically synthesized and transfected into U87 cells. The apoptosis, proliferation, and invasion of the cells were evaluated. The relationship between miR-21 and PTEN or caspase was identified by bioinformatics and Western blot.</p><p><b>RESULTS</b>Inhibiting miR-21 expression led to U87 cell growth suppression, apoptosis induction, invasion reduction, caspase-3 activity elevation and caspase-9 activation, but did not affect PTEN and caspase-8 expression.</p><p><b>CONCLUSION</b>miR-21 may function as an antiapoptotic miRNA in U87 cells. Inhibiting miR-21 expression could induce U87 cell apoptosis via caspase-9 and 3 activation, but not PTEN activation.</p>

¿Los ARNs antisentido cumplen algún rol en eucariontes? / Does antisense RNA have any role in eukaryotes ?

The presence of antisense transcripts has been described in a wide variety of eucaryotic organisms, with a diversity of implied biological functions in development, control of cell cycles, hormonal responses, control of proliferation, structure, viral replication, and others. Reports have suggested that this type of control may operate at different levels of genic expression, whether in transcription, maturation, transport, stability, or translation of a given trascript. It is evident that the expression of antisense RNA has true importance in Eukaryotes, as already established in prokaryotic organisms. The objective of the present review is to present the advances in the mechanics of regulation, and roles already established for the expressions of antisense RNA in eukaryotic organisms.

La presencia de transcritos antisentidos ha sido descrita en una amplia variedad de organismos eucariontes, implicados en una diversidad de funciones biológicas como el desarrollo, el control del ciclo celular, respuesta hormonal, control de la proliferación, estructura, replicación viral, etc. Los informes indican que este control puede darse a diversos niveles de la expresión génica, ya sea en la transcripción, maduración, transporte, estabilidad y traducción de un determinado transcrito. Es evidente que la expresión del ARN antisentido tiene una real importancia en eucariontes, como ya ha sido establecido en organismos procariontes. Esta revisión tiene como objetivo mostrar los avances en los mecanismos de regulación y roles ya establecidos de la expresión de ARN antisentidos en organismos eucariontes.

Calcium sensing receptor forms complex with and is up-regulated by caveolin-1 in cultured human osteosarcoma (Saos-2) cells

The calcium sensing receptor (CaSR) plays an important role for sensing local changes in the extracellular calcium concentration ([Ca2+]o) in bone remodeling. Although the function of CaSR is known, the regulatory mechanism of CaSR remains controversial. We report here the regulatory effect of caveolin on CaSR function as a process of CaSR regulation by using the human osteosarcoma cell line (Saos-2). The intracellular calcium concentration ([Ca2+]i) was increased by an increment of [Ca2+]o. This [Ca2+]i increment was inhibited by the pretreatment with NPS 2390, an antagonist of CaSR. RT-PCR and Western blot analysis of Saos-2 cells revealed the presence of CaSR, caveolin (Cav)-1 and -2 in both mRNA and protein expressions, but there was no expression of Cav-3 mRNA and protein in the cells. In the isolated caveolae-rich membrane fraction from Saos-2 cells, the CaSR, Cav-1 and Cav-2 proteins were localized in same fractions (fraction number 4 and 5). The immuno-precipitation experiment using the respective antibodies showed complex formation between the CaSR and Cav-1, but no complex formation of CaSR and Cav-2. Confocal microscopy also supported the co-localization of CaSR and Cav-1 at the plasma membrane. Functionally, the [Ca2+]o- induced [Ca2+]i increment was attenuated by the introduction of Cav-1 antisense oligodeoxynucleotide (ODN). From these results, in Saos-2 cells, the function of CaSR might be regulated by binding with Cav-1. Considering the decrement of CaSR activity by antisense ODN, Cav-1 up-regulates the function of CaSR under normal physiological conditions, and it may play an important role in the diverse pathophysiological processes of bone remodeling or in the CaSR- related disorders in the body.

Expression of bFGF and VEGF in acute leukemia and its effects on HL-60 cell growth / 中国实验血液学杂志

The study was to investigate the expression levels of basic fibroblast growth factor (bFGF) and vascular endothelial growth factor (VEGF) in the serum of patients with acute leukemia and supernatants of leukemia cell lines as well as effects of VEGF-specific antisense oligodeoxynucleotides (ASODN) on the growth of HL-60 cells. Meanwhile the methods to evaluate the VEGF level in the serum of patients with acute leukemia were explored. The levels of bFGF and VEGF in the serum from 32 patients with acute leukemia and 10 healthy subjects and in the supernatants of 5 various human leukemia cell lines were quantified by means of the enzyme-linked immunosorbent assay (ELISA) and were compared. VEGF levels were evaluated not only without standardization but also after standardized by platelet and finally expressed as VEGF/PLT (pg/10(6)). After with different concentrations of VEGF ASODN, HL-60 cell viability was examined with MTT assay and VEGF levels in supernatants were measured with ELISA, respectively. The results showed that bFGF was detected (3 pg/ml) in 14 out of 32 serum samples from patients with acute leukemia, and the positive (37.5%) was significantly higher than that in healthy controls (10%) (P < 0.01). 3 out of 5 supernanant samples obtained from leukemia cell lines demonstrated positive for bFGF as well. There is no difference of the serum VEGF levels between leukemia patients and healthy controls, but the serum VEGF levels in the serum from leukemia patients were significantly higher than those in healthy controls (P < 0.05) after standardization. 4 out of 5 leukemia cell (U937 excluded) were found to express VEGF in the supernanant. After exposure of HL-60 cells to VEGF ASODN at a concentration of 0.5, 1 and 5 micromol/L for 24 hours, the cell viability gradually dropped down to lower levels (P < 0.05 vs controls). After treatment of HL-60 cells with VEGF ASODN at a concentration of 1, 5 and 20 micromol/L for 24 hours, the VEGF levels in supernatants of target cells decreased (P < 0.05 vs controls). The patients with acute leukemia represented the higher levels of serum bFGF and VEGF than controls. Most of leukemia cell lines expressed bFGF and VEGF at different levels. It is concluded that bFGF and VEGF both have effects on regulations of angiogenesis in acute leukemia, but VEGF plays a pivotal role. VEGF-specific ASODN may have a role in them VEGF expression downregulated. Different results may be obtained in the evaluation of VEGF levels in the serum of patients with acute leukemia if different calculation methods are used. The methods reported can measure leukemia associated VEGF more accurately.

Effects of dentin sialophosphoprotein antisense oligodeoxynucleotide on ultrastructure of mouse tooth germ / 华西口腔医学杂志

<p><b>OBJECTIVE</b>To investigate more deeply the function and mechanism of DSPP during tooth development.</p><p><b>METHODS</b>Explants of tooth germs from embryonic 17th day mice were divided into two groups. In the control experiment, explants were cultured in agarose semi-solid medium under serum-free and chemically defined conditions, while explants in the other group were cultured with 30 mumol/L, 15 bp antisense oligodeoxynucleotide targeted to DSPP mRNA. After 10 ds, the explants were examined by transmission electron microscope. The width of dentin matrix at the tip of the cusps were then measured and statistically analyzed with Student t-test.</p><p><b>RESULTS</b>Ultrastructure analyses showed that large cisternae of the rough endoplasmic reticulum (RER) existed in the odontoblasts at the tip of the cusps of antisense-treated explants and the average thickness of dentin matrix (2.5 microns) was thinner compared to the control ones (3 microns, P < 0.001). In addition, the collagen fibers in extracellular matrix were disorganized.</p><p><b>CONCLUSION</b>These findings indicated that DSPP played an important role in keeping tooth normal development, as well as in dentin mineralization by maintaining odontoblasts' secreting ability and controlling fiber structure and orientation.</p>