Induction function of siRNA-mediated survivin gene silencing on nasopharyngeal carcinoma cell apoptosis.

We examined the function of survivin gene expression in patients with nasopharyngeal carcinoma (NPC), as well as small interfering RNA (siRNA) on controlling CNE-2 NPC proliferation and apoptosis. Immunohistological methods, in situ hybridization, and reverse transcription-polymerase chain reaction technique were used to detect survivin protein and mRNA expression. We designed an siRNA sequence to inhibit survivin gene expression. The MTT method was used to examine the function of siRNA on controlling cell growth and proliferation. Induction of cell apoptosis by siRNA was examined by flow cytometry; electron microscopy was used to observe ultrastructure changes in CNE-2 cells. Western blotting was used to detect survivin gene expression. The survivin protein was expressed in 71.9% of cells, while its mRNA was expressed in 65.6% of cells. Relative mRNA expression was 4.16 x 10(-2); these data for the control groups were 23.3, 33.3, and 4.42 x 10(-4), respectively. Following transfection with 3 different siRNA sequences, survivin mRNA expression in CNE-2 cells was decreased. Inhibition of cell proliferation and rate of apoptosis increased with increasing siRNA concentration. Western blotting revealed decreased survivin expression and electron microscopy revealed ultrastructural changes in cancer cells. Survivin gene expression in NPC generally increased. In vitro transcription of siRNA decreased CNE-2 survivin gene expression, and different sequences of siRNA decrease gene expression in CNE-2 cells to varying degrees. Transfected siRNA3 can effectively inhibit CNE-2 cell proliferation and induce apoptosis; gene silencing using siRNA may represent a new treatment for NPC.

Effect of epristeride on expression of TGF-beta receptor in rat prostatic epithelial cells in vitro.

AIM: To assess the effect of epristeride on the expression of transforming growth factor beta type II receptor (TbetaR II) in rat prostatic epithelial cells in vitro. METHODS: RT-PCR and Western blot were used to quantitatively detect the mRNA and protein expressions of TbetaR II in rat prostatic epithelial cells treated or untreated with epristeride. Immunocytochemical staining method was used to qualitatively analyze the expression of TbetaR II protein. RESULTS: After treatment with epristeride 180 or 360 nmol/L, TbetaR II mRNA expression levels were 0.56 +/- 0.08 and 0.59 +/- 0.07, respectively, which were significantly up-regulated compared with control cells (0.38 +/- 0.04, P < 0.05); expression level of TbetaR II protein were 3163 +/- 920 and 6769 +/- 1941, respectively, which were also markedly up-regulated compared with control cells (536 +/- 240, P < 0.05). Immunostaining showed weak positive reaction in control cells, while strong staining of TbetaR II was found in epristeride-treated cells. CONCLUSION: Epristeride may up-regulate the expression of TbetaR II to induce apoptosis of prostatic epithelial cells.

Inhibition of cultured rat prostatic epithelial cell growth by epristeride in vitro.

AIM: To study the molecular mechanism of rat prostate atrophy induced by epristeride. METHODS: MTT test was used to determine the effect of epristeride on the growth of prostatic epithelial cell induced by exogenous epithelial growth factor (EGF) or insulin-like growth factor-I (IGF-I). RT-PCR and flow cytometry were then used to quantitatively detect the mRNA and protein expressions of EGFR and IGF-I R of the epithelial cells treated or untreated with epristeride. RESULTS: Epristeride attenuated growth of epithelial cells induced by exogenous EGF, IGF-I. Epristeride 360 nmol/L inhibited EGFR and IGF-I R expression at mRNA level, while epristeride 180 nmol/L had no marked effect on EGFR and IGF-I R mRNA expression. Both epristeride 180 nmol/L and 360 nmol/L could down regulate EGFR and IGF-I R protein levels. CONCLUSION: The molecular mechanisms of prostatic epithelial cell atrophy induced by epristeride might be associated with alteration in the expression of growth factor receptors such as EGF and IGF-I.

Atrophy and apoptosis in ventral prostate of rats induced by 5alpha-reductase inhibitor, epristeride.

AIM: To study molecular mechanism of epristeride in the treatment of benign prostatic hyperplasia and discuss the possibility of using prostate acid phosphatase (ACP) as a marker of the atrophy of prostatic gland in vivo. METHODS: Morphological changes in cells were observed by light microscope. TdT-mediated dUTP-biotin nick end labeling (TUNEL) technique and agarose gel electrophoresis were performed to detect the nucleosomal DNA fragmentation. The activity of pACP was also assayed. RESULTS: Apoptosis occurred in both castration- and epristeride- treatment group. Both the degree and extent of apoptosis are much larger in the group of castration than that of epristeride-treated group. Both epristeride and castration decreased the prostate wet weight and DNA content but increased the prostate DNA concentration. Maximal or near maximal decreases were seen by d 10 in both groups. The activity of ACP was decreased by both castration and epristeride treatment. Changes in the ACP activity during treatment were coincide with other changes such as the prostate wet weight and DNA content. CONCLUSION: Apoptosis induced by epristeride was one of mechanisms in the treatment of benign prostatic hyperplasia and the activity of ACP could be used as a marker of prostate atrophy.

Knockdown of IGF-IR by Antisense Oligodeoxynucleotide auguments the sensitivity of bladder cancer cells to mitomycin.

AIM: To investigate whether insulin-like growth factor I receptor (IGF-IR) was involved in drug resistance of bladder cancer cells. METHODS: RT-PCR was used to detect the mRNA expression of IGF-I, IGF-II, and IGF-IR in T24 cells and normal urothelial cells. Flow cytometry and MTT tests were used to assess the effect of antisense oligodeoxynucleotide (ODN) on drug sensitivities and apoptosis of T24 cells to mitomycin (MMC). Western blot was used to analyze the effect of ODN on expression of IGF-I R protein. RESULTS: mRNA of IGF-I, IGF-II, and IGF-IR were strongly expressed in serum-free cultured T24 cell line, whereas normal urothelial cells did not express these factors/receptors or only in trace levels; knockdown of IGF-IR by antisense ODN greatly inhibited the growth of bladder cancer cells and enhanced sensitivity and apoptosis of T24 cells to MMC. CONCLUSION: These results suggested that blockage of IGF-IR signaling might potentially contribute to the treatment of bladder cancer cells which were insensitive to chemotherapy.

Inhibition of regrowth of prostatic glandular cells by epristeride.

AIM: To evaluate the ability of epristeride to inhibit the prostatic glandular regrowth. METHODS: Normal rats were castrated. Testosterone was injected to induce the regrowth of glandular cells. HE staining was performed. The height of the glandular epithelium and the acinar luminal areas were determined, and dihydrotestosterone (DHT) was detected by immunohistochemistry. RESULTS: Both the height and the acinar luminal areas were reduced by 48 % and 55 % in epristeride-treated group compared with control group respectively. The staining of DHT was comparatively strong in the control group. After 30-d of treatment, it turned much weaker. CONCLUSION: The regrowth of glandular cells was inhibited by epristeride via declining of the DHT concentration in the rat prostate.

Effect of epristeride on the expression of IGF-1 and TGF-beta receptors in androgen-induced castrated rat prostate.

The development of benign prostatic hyperplasia (BPH) is an androgen-dependent process that may be mediated by a number of locally produced growth factors. Among them, insulin-like growth factor 1 (IGF-1) and transforming growth factor beta (TGF beta) are thought important in regulating prostate growth and homeostasis, and their expression undergoes changes in proliferative prostatic disease. Epristeride, a 5 alpha-reductase inhibitor, is an effective drug in the treatment of BPH, inducing regressive changes in the prostate. This study was designed to assess the effects of epristeride on expression of these two factors at mRNA and protein levels in castrated rats maintained with exogenous testosterone. Epristeride treatment caused significant reduction in ventral prostate weight in a dose-dependent manner. There was a positive correlation between IGF-1 mRNA expression and ventral prostate weight and an inverse correlation between TGF-beta 1 mRNA expression and ventral prostate weight. Immunohistochemistry showed strong IGF-1 receptor immunoreactivity in the prostatic epithelial cells of untreated animals. In situ hybridization demonstrated high levels of IGF-1 mRNA expression both in the prostatic stromal and epithelial cells of untreated rats. In treated rats, both IGF-1 receptor protein and IGF-1 mRNA levels decreased significantly, and IGF-1 mRNA was mainly expressed in prostatic stromal cells. Weak expression of TGF beta receptors at the protein level and TGF beta at the mRNA level were found in the prostatic hyperplastic epithelial cells of untreated rats. In treated animals, intense T beta RII immunoreactivity was observed in epithelial cells, and a higher level of TGF beta mRNA was observed in both epithelial cells and stromal cells compared with control animals. In our opinion, the effect of epristeride on rat prostatic atrophy might be mediated via local growth factor(s).

Blockage of IGF-1R signaling sensitizes urinary bladder cancer cells to mitomycin-mediated cytotoxicity.

A major problem which is poorly understood in the management of bladder cancer is low sensitivity to chemotherapy and high recurrence after transurethral resection. Insulin-like growth factor 1 receptor (IGF-1R) signaling plays a very important role in progression, invasion and metastasis of bladder cancer cells. In this study, we investigated whether IGF-1R was involved in the growth stimulating activity and drug resistance of bladder cancer cells. The results showed: The mRNAs of IGF-1, IGF-2 and IGF-1R were strongly expressed in serum-free cultured T24 cell line, whereas normal urothelial cells did not express these factors/receptors or only in trace levels; T24 cell responded far better to growth stimulation by IGF-1 than did normal urothelial cells; blockage of IGF1R by antisense oligodeoxynucleotide (ODN) significantly inhibited the growth of T24 cell and enhanced sensitivity and apoptosis of T24 cells to mitomycin (MMC). These results suggested that blockage of IGF-IR signaling might potentially contribute to the treatment of bladder cancer cells which are insensitive to chemotherapy.

Moclobemide-induced gynecomastia in rats.

AIM: To study the toxic effect of moclobemide on male breast and to elucidate its mechanism of action. METHODS: Routine histopathological analysis was used to diagnose the effect of moclobemide on male breast in rats. Plasma concentrations of estrogen, androgen, and prolactin were measured by a ratioimmunometer and relative receptors of mammary gland tissue were detected immunohistochemically. RESULTS: After 180-d moclobemide treatment, the presence of gynecomastia was 0, 5, 5, 7/10 rats in 0, 60, 240, and 600 mg/kg groups, respectively. After 30-d convalescence, only one rat in 600 mg/kg group got the incidence of gynecomastia. Serum prolactin concentration had a trend to decrease with increasing dose and prolactin receptors in mammary gland were up-regulated. CONCLUSION: Long-term treatment with moclobemide causes gynecomastia in rats, which is reversible. The mechanism of moclobemide-induced gynecomastia may be related to the increase in prolactin receptors in mammary glands.

The mechanism of epristeride against benign prostatic hyperplasia.

Epristeride, a 5alpha-reductase inhibitor, decreases prostate size and improves symptoms in men with benign prostatic hyperplasia. However, little is known about the histopathology of the prostate after treatment with epristeride. To study the relationship between apoptosis and the mechanism of epristeride in the treatment of benign prostatic hyperplasia, the induction of apoptosis by epristeride was detected and measured in vitro by: (a) observing morphological changes in cells by light microscopy; (b) comparing the relative content of dihydrotestosterone in the rat prostate epithelial cells untreated and treated with epristeride by microspectrophotometry; (c) estimating changes in cell size and DNA integrity by flow cytometry; and (d) monitoring nucleosomal DNA fragmentation by agarose gel electrophoresis. The cells treated with epristeride showed a reduction in cell size, an increase in the cytoplasm/nuclear ratio, which is indicative of the condensation of nuclear chromatin, a significant decrease in optical density at 580 nm (OD580 nm), and an oligonucleosomal ladder and a subdiploid peak of DNA characteristic of apoptosis. Therefore, the mechanism of epristeride in the treatment of benign prostatic hyperplasia might be apoptosis stimulated by decreasing dihydrotestosterone level.

Reversible long-term toxicity of epristeride in beagle dogs.

Epristeride (17beta-N-t-butylcarboxamide-androst-3, 5-diene-3-carboxylic acid) is an uncompetitive inhibitor of steroid 5alpha-reductase, the enzyme that converts testosterone to dihydrotestosterone (DHT), and has been shown to retard the growth of hyperplastic prostates. The objective of the current investigation was to research the toxic effects of epristeride and to demonstrate its reversible. In the experiment, 18 beagle dogs (male, about 6 months old) were used and divided into six groups, with each group containing three dogs. Groups A and B were placebo-treated for 180 and 240 days, Groups C and D were treated with 10 and 100 mg/kg epristeride for 180 days, and Groups E and F were treated with 10 and 100 mg/kg epristeride for 180 days and then were placebo-treated for 60 days (total 240 days), respectively. Routine analyses were performed at the 1st, 30th, 90th, 180th, and 240th days, and the dogs were autopsied at the 180th or 240th day for systemic examination and measured for relative DNA content in single prostatic epithelial cells. Each prostate was fixed with 4% Formalin, embedded in paraffin, sectioned at 6 micron, and immunohistochemically stained for assaying the relative content (transmittance) of prostatic specific antigen (PSA) and DHT (%) with a microspectrophotometer at 650-nm wavelength. The results were that 180 days of toxicity with epristeride (100 mg/kg) on interstitial cells of testes and DNA in prostatic epithelial cells couldn't reverse during 60 days of convalescence and that the DHT and PSA levels in the gland, the volume of the gland, glandular epithelial cell height, and acinar luminal area could reverse to normal during the same convalescence. To our knowledge this is the first study documenting the toxicity of epristeride. It is necessary to further study the molecular and clinical toxicity of epristeride.

Comet electrophoresis of blood nucleated cells in genotoxicity assessment.

AIM: Genotoxicity evaluations of several different chemicals including L-4-oxalysine, 10-Hydroxycamptothecin (HCT), 19-norprogesteron (ST1435), dimethyl sulfoxide (Me2SO), bleomycin (BLM), and mitomycin C (MMC). METHODS: Alkaline comet assay in vitro (single cell gel) (SCG). RESULTS: L-4-oxalysine and HCT did not cause directly DNA damage. ST1435, the subdermal implant progestin, had no effect on DNA damage until the dose level up to 4 mmol.L-1. Me2SO did not increase DNA damage at concentration below 2%, but showed a concentration-dependent DNA damage at > or = 4%. Bleomycin and mitomycin C demonstrated a strong dose-dependent DNA damage. CONCLUSION: Comet assay as a tool to test the genotoxicity of suspected chemicals, is rapid, simple, sensitive, good reproducible, and inexpensive.

A novel in vitro model to screen steroid 5 alpha-reductase inhibitors against benign prostatic hyperplasia.

A convenient and rapid in vitro model to screen steroid 5 alpha-reductase inhibitors, which are effective in the treatment of benign prostatic hyperplasia (BPH), was developed. In the presence of nicotinamide adenine dinucleotide phosphate (NADPH), steroid 5 alpha-reductase converts testosterone to dihydrotestosterone (DHT) which is a major etiologic factor of BPH. NADPH has characteristic absorbance at 340 nm, and the absorbance spectrum may be used to identify NADPH as a kind of the substrate in this enzymatic reaction. In this paper, NADPH, steroid 5 alpha-reductase, series concentration of testosterone and finasteride, and 4 ml Tris-HCl buffer were continuously incubated together at 37 degrees C and the NADPH OD values were continually measured. The descending rate of NADPH OD340nm value by linear regression from the beginning to the 10th minute is close to the initial velocity of the enzymatic reaction. The precise activity of the steroid 5 alpha-reductase was the slope after subtracting that of the blank control. The inhibition constant (Ki) of steroid 5 alpha-reductase inhibitors could be calculated according to the Lineweaver-Burk plots. Two drug screening models, the most common isotope model and the novel model, were compared in this paper. The result showed that the latter one is more economical, quicker and more effective than the former one.

Mutagenicity tests on epristeride in vitro and in vivo.

AIM: To evaluate the genetic effects of epristeride (Epr), a new prospective drug for treating benign prostatic hyperplasia. METHODS: 1) Assaying reverse mutation in histidine nutritional deficiency strain of Salmonella typhimurium 2) detecting chromosome aberrations in Chinese hamster lung cells (CHL); 3) micronucleus assays of mouse bone marrow; 4) counting sperm shape abnormalities 35 d after first ig Epr. RESULTS: 1) The reverse mutation happened at almost the same rate of the negative control. Epr did not induce bacterial mutation. 2) In vitro, the rates of aberration were all below 3%, thus Epr did not induce chromosome damage in CHL. 3) Micronucleated polychromatic erythroblasts (PCE) were not apparently more than those of sovent control, Epr did not induce the formation of micronuclei in PCE. 4) With Epr 818, 682, and 341 mg.kg-1, the head abnormalities of sperms were 5.3% +/- 2.7%, 5.3% +/- 1.9%, and 5.2% +/- 1.2%, respectively. CONCLUSION: No genetic toxicity of Epr was detected.

Toxicity to transferred rat embryos after aspirin treatment during preimplantation stage in vivo.

AIM: To explore the relationship between drug-induced blastopathies and post-implantation embryotoxicity or developmental defects. METHODS: Pregnant rats on d 3 were given intragastrically aspirin (0.25, 0.5, and 1 g.kg-1). On d 4, the blastocysts were transferred into the uterine horns of pseudopregnant rats (made by mating with male rats which had been given intragastrically 3-chloro-1,2-propanediol 5 mg.kg-1 for 5 d). Uterine contents were examined at term. RESULTS: The frequency of blastocysts with morphological alterations (FBMA) was increased on d 4 of gestation. The implantation rate was lower than that of the controls. A dose-related increase in resorption (55.2%, 69.5%, and 85.2%) and malformation rate (3.8%, 44.4%, and 25%), and decrease in viability rate of fetuses (44.8%, 30.5%, and 14.8%) were observed in test groups with correlations to FBMA. CONCLUSION: Embryotoxicity and fetal malformations were induced by treatment of aspirin before implantation in a dose-dependent manner.

[Leukemia associated with bimkolane].

Psoriatic patients and mice treated with bimolane were observed. The frequency of chromosomes aberration and micro-nuclear cells presence in the treated group (11 cases) was significantly higher than that in the control group (11 cases, P < 0.005; < 0.001). Study of lymphocytic subsets showed that value of CD4/CD8 in the peripheral blood of the treated group was lower than that of the control group (P < 0.05). The level of serum IgM in the treated group was also lower (P < 0.05). There were 10 mice suffering from leukemia (7 mice with acute promyelocytic leukemia) in a treated group of 40 mice of an inbred line of 615 mice, while there was no leukemia in a control group of 20 mice of the same species. The morbidity of leukemia in the treated mice was higher than that of controls (P < 0.05).

[Lack of mutagenicity and teratogenicity of 16-methylene-17 alpha-acetoxy-19-norprogesterone].

16-Methylene-17 alpha-acetoxy-19-norprogesterone (ST-1435) is a new antifertility agent. ST-1435 silastic capsule was implanted sc in the nuchal region of rats on d 6 of gestation at 75, 300, and 600 mg.kg-1. The rats were killed on d 20. In comparison with the control, the treated groups showed no significant differences in maternal body weights, number of corpora lutea, and the development of embryos and fetuses. The number of dead fetuses decreased and live fetuses increased slightly. ST-1435 did not affect the frequency of micronucleated polychromatic erythrocytes, nor induce chromosomal aberrations in cultured CHL cells, or increase the revertants of Salmonella typhimurium TA97, TA98, TA100, and TA102 in Ames test. The results suggested that ST-1435 had neither teratogenicity nor mutagenicity.

[Effects of 10-hydroxycamptothecin on induced chromosome aberrations in Chinese hamster ovary cells and micronuclei in mouse bone marrow and fetal liver].

10-Hydroxycamptothecin (HC) is a new antitumor principle isolated from Camptotheca acuminata indigenous to China. The genetic toxicity of HC was assessed by mouse bone marrow and transplacental micronucleus test as well as Chinese hamster ovary cell chromosomal aberrations. All of these tests showed positive results. The highest rate of chromosomal aberrations was 83% at 0.125 microgram/ml for 48 h. The number of micronucleated polychromatic erythrocytes in bone marrow of mice was remarkably increased in 19.8% cells at 12.5 mg/kg for 24 h. The micronucleus formation was most often seen at 16 h after im HC in fetal liver and 24 h in maternal bone marrow. The peaks were 36 +/- 19 and 31 +/- 10%, respectively. The results from in vivo and in vitro suggest HC is a mutagen, furthermore, a transplacental mutagen in mouse.

Anti-troponin-T monoclonal antibody crossreacts with all muscle types.

Monoclonal antibodies to troponin-T were produced by the hybridoma technique. Culture supernatants were initially screened using an enzyme-linked immunoabsorbent assay (ELISA). Positive clones were subcloned twice and further characterized. One of these, 7/H3:C9:D10, produced antibodies against troponin-T; immunoblotting experiments indicated its specificity for only troponin-T when challenged with a variety of striated muscle myofibrillar proteins. Indirect immunofluorescence staining with the antibody shows specific I-band staining in both adult and embryonic skeletal and cardiac muscle of various vertebrate species. In addition, intense but diffuse cytoplasmic staining was seen in chicken gizzard smooth muscle. Our results suggest that troponin-T contains an antigenic determinant that is common to both striated and smooth muscle.