Anti-proliferation effects of isorhamnetin on lung cancer cells in vitro and in vivo.

BACKGROUND: Isorhamnetin (Iso), a novel and essential monomer derived from total flavones of Hippophae rhamnoides that has long been used as a traditional Chinese medicine for angina pectoris and acute myocardial infarction, has also shown a spectrum of antitumor activity. However, little is known about the mechanisms of action Iso on cancer cells. OBJECTIVES: To investigate the effects of Iso on A549 lung cancer cells and underlying mechanisms. MATERIALS AND METHODS: A549 cells were treated with 10~320 µg/ml Iso. Their morphological and cellular characteristics were assessed by light and electronic microscopy. Growth inhibition was analyzed by MTT, clonogenic and growth curve assays. Apoptotic characteristics of cells were determined by flow cytometry (FCM), DNA fragmentation, single cell gel electrophoresis (comet) assay, immunocytochemistry and terminal deoxynucleotidyl transferase nick end labeling (TUNEL) . Tumor models were setup by transplanting Lewis lung carcinoma cells into C57BL/6 mice, and the weights and sizes of tumors were measured. RESULTS: Iso markedly inhibited the growth of A549 cells with induction of apoptotic changes. Iso at 20 µg/ml, could induce A549 cell apoptosis, up-regulate the expression of apoptosis genes Bax, Caspase-3 and P53, and down-regulate the expression of Bcl-2, cyclinD1 and PCNA protein. The tumors in tumor-bearing mice treated with Iso were significantly smaller than in the control group. The results of apoptosis-related genes, PCNA, cyclinD1 and other protein expression levels of transplanted Lewis cells were the same as those of A549 cells in vitro. CONCLUSIONS: Iso, a natural single compound isolated from total flavones, has antiproliferative activity against lung cancer in vitro and in vivo. Its mechanisms of action may involve apoptosis of cells induced by down-regulation of oncogenes and up-regulation of apoptotic genes.

No association between hypertension and risk for Alzheimer's disease: a meta-analysis of longitudinal studies.

This study examined the association between hypertension and AD by using a quantitative meta-analysis of longitudinal studies. EMBASE and MEDLINE were searched for articles published up to February 2011. All studies that examined the association of hypertension or antihypertensive medication use with the onset of AD were included. Pooled relative risks (RR) were calculated using fixed and random effects models. Twelve studies met our inclusion criteria for this meta-analysis. All subjects were without dementia at baseline. Among them, 9 studies compared the incidence of AD between subjects with (7,270) and without (8,022) hypertension. The quantitative meta-analysis showed that there was no significant difference in incidence of AD (RR: 1.02, 95% confidence interval (CI): 0.91-1.14) between subjects with and without hypertension. Seven studies compared the incidence of AD between subjects with (8,703) and without (13,041) antihypertensive medication use. The quantitative meta-analysis showed that there was no significant difference in incidence of AD (RR: 0.90, 95% CI: 0.79-1.03) between subjects with and without antihypertensive medication use. The quantitative meta-analysis showed that neither hypertension nor antihypertensive medication use was associated with risk for incident AD.

Gene therapy with recombinant adenovirus encoding endostatin encapsulated in cationic liposome in coxsackievirus and adenovirus receptor-deficient colon carcinoma murine models.

Adenovirus (Ad)-based antiangiogenesis gene therapy is a promising approach for cancer treatment. Downregulation or loss of coxsackievirus and adenovirus receptor (CAR) is often detected in various human cancers, which hampers adenoviral gene therapy approaches. Cationic liposome-complexed adenoviral vectors have been proven useful in CAR-deficient cells to enhance therapeutic gene transfer in vivo. Here, we investigated the antitumor effects of recombinant adenovirus encoding endostatin (Ad-hE) encapsulated in cationic liposome (Ad-hE/Lipo) on CAR-deficient CT26 colon carcinoma murine models. In vitro, Ad-hE/Lipo enhanced adenovirus transfection in CAR-deficient cells (CT26), and endostatin gene expression was measured by both qualitative and quantitative detection. In addition, an antibody neutralizing assay indicated that neutralizing serum inhibited naked adenovirus 5 (Ad5) at rather higher dilution than the complexes of Ad5 and cationic liposomes (Ad5-CL), which demonstrated that Ad5-CL was more capable of protecting Ad5 from neutralization. In vivo, Ad-hE/Lipo treatment in the murine CT26 tumor model by intratumoral injection resulted in marked suppression of tumor growth and prolonged survival time, which was associated with a decreased number of microvessels and increased apoptosis of tumor cells. In conclusion, recombinant endostatin adenovirus encapsulated with cationic liposome effectively inhibited CAR-deficient tumor growth through an antiangiogenic mechanism in murine models without marked toxicity, thus showing a feasible strategy for clinical applications.

[Relationship between polymorphism of hypoxia inducible factor-1alpha and cervical cancer in Han population in Sichuan Province of China].

OBJECTIVE: To investigate the relationship between single nucleotide polymorphism (SNP) of hypoxia inducible factor-1alpha (HIF-1alpha) C1772T and genetic susceptibility to and clinical-pathological features of cervical cancers in Han population in Sichuan province of China. METHODS: A case control study was undertaken in Sichuan province of China, with 97 patients with uterine cervical cancer as case group and 117 negative for intraepithelial lesion or malignancy (NILM) patients as control group. Their gene types in HIF-1alpha C1772T were identified with a combination of polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP). RESULTS: The distribution of the frequencies of T/T, T/C and C/C genotypes in the two groups differed significantly (P < 0.01); T allele frequency in the patients with cervical cancer was much higher than that in the controls (P < 0.01). There were no significant differences in the distributions of T/T, T/C and C/C genotypes among cervical cancer patients at different FIGO stages, pathological grading, stromal invasive depth, lymph node metastasis and vascular invasions (P > 0.05). CONCLUSION: T/C and T/T genotypes of HIF-1alpha C1772T are genetic susceptibility factors for cervical cancer in Han population in Sichuan province of China. HIF-1alpha C1772T SNP probably has no relationship with clinical-pathological features of cervical cancer.

[Influence of heart rate's circadian rhythm in postoperation of intracerebral hemorrhage with different surgery].

OBJECTIVE: To study the influence of different surgery on heart rate's circadian rhythm in postoperation of intracerebral hemorrhage. METHODS: One hundren cases of hypertensive intracerebral hemorrhage at basal ganglia had been collected. According to methods of operation, cases were divided into two groups. Forty-three cases and 57 cases were treated respectively by standard craniotomy (Craniotomy group) and microinvasion of puncturation (Microinvasion group) to remove hematoma. Value of heart rate (HR) was recorded by monitor every 1 hour for 9 days. Circadian rhythm of every day was analyzed by single cosinor and population mean-cosinor. RESULTS: Circadian rhythm of postoperative HR appeared (P < 0.001) in microinvasion group after operation. Circadian rhythm of postoperative HR had not been appearing (P < 0.001) in craniotomy group until to the 8th day. All amplitudes in microinvasion group are higher than that in craniotomy group. Both of amplitudes in two groups begin to be low during 4-7 d after operation and recover the level till the 8th day. CONCLUSION: Influence of circadian rhythm in craniotomy was more severe than microinvasion puncturation. There would be a intimate relationship between encephaledema and recovery of circadian rhythm after neurosurgery.

[Effects of circadian gene mPer1 on therapeutic sensitivity of adriamycin].

OBJECTIVE: To study the effects of mPer1 gene on the response of mammary carcinoma EMT6 cells to Adriamycin in vitro. METHODS: The eukaryotic expression vector pcDNA3. 1 (+)-mPer1 was transfected into the EMT6 cells (EMT6-mPerl). The vector pcDNA3. 1(+) transfect was also performed to serve as the control (EMT6-vect). The transfect efficiency was detected by RT-PCR and Western Blotting. The transfect cells were treated with Adriamycin in vitro. The apoptosis and distribution of cells in the cell cycle were analysed by FCM. The cell proliferation was detected by MTT assay. RT-PCR was used to show the mRNA expression of apoptosis-related genes. RESULTS: The mPerl-transfected EMT6 cells revealed S phase arrest, increased rate of apoptosis [EMT6-vect: (65.65 +/- 0.07)%; EMT6-mPer1: (72.35 +/- 0.57)%], decreased cell proliferation CEMT6-vect: (42.18 +/- 5.73)%; EMT6-mPer1: (53.28 +/- 7.32%)%] and stronger expression of p53 mRNA in RT-PCR (EMT6-vect, 0.48 +/- 0.08; EMT6-mPer1: 1.18 +/- 0.02). CONCLUSION: mPer1 gene can improve the drug sensitivity of this cell line to ADM in vitro.

[The influence of circadian clock gene on mouse sperm fertility in RNAi].

OBJECTIVE: To study the influence of circadian Clock gene on the fertilization ability of sperm via attenuating the expression of Clock with RNAi in male mice. METHODS: After the injection of RNAi plasmid into mice testes, the expression of circadian Clock gene of testis tissue was determined by western Blotting. The fertilization ability of sperm was evaluated by various indices, including litter size in mated female mice, sperm count, sperm motility, in vitro fertilization (IVF) rate and acrosome development. RESULTS: The RNAi plasmid targeting circadian gene Clock attenuated the expression of CLOCK in mice testis, reduced in vitro fertilization CIVF) rate and the conception rate in viva, and also decreased sperm acrosin activity. CONCLUSION: Circadian gene Clock is related to the reproductive function in male mice. It probably affect sperm fertilization ability by regulating sperm acrosin activity.

[Identification of RACK1-PER1 protein interaction sites].

OBJECTIVE: By screening the cDNA library of suprachiasmatic nucleus (SCN) region of human hypothalamus, we try to capture the novel proteins interacting with PER1 and investigate the interplaying characteristic of RACK1 and PER1. METHODS: By yeast two-hybrid system, the new protein in human SCN, which was associating with PER1-PAS domain was obtained. Then five truncated fragments of RACK1 cDNA was cloned into yeast expression vector to form recombinant library plasmid and was co-transformed into yeast strain AH109 with bait plasmid containing PER1-PAS domain. The transformants were selected via nutrition-deficient medium, and the positive clones were identified or obtained by checking the expressin of report gene. At last all the protein interactions were confirmed by co-immunoprecipitation tests. RESULTS: one of the positive clones in human SCN cDNA library were identified with part of the RACK1 protein sequence. Three positive clones, which contained respectively the fragment of RACK1 (WD1-7), RACK1 (WD4-7) or RACK1 (WD5-7) were obtained through yeast two-hybrid screen with various RACK1 fragments and PER1-PAS domain. The RACK1 and PER1 protein interaction was determined by beta-galactosidase assay and co-immunopreipitation. CONCLUSION: The direct interaction between RACK1 and PER1 has been approved. RACK1 is composed of seven WD40 repeats and the minimal interacting sites are limited in V-VII WD40 domains in the present study,which means C-terminal amino acid of RACK1 may be essential to the interacting.

[Inhibitory effects of deoxyribozyme on the expression of Period1 gene in vitro and on morphine-induced psychic dependence in mice].

OBJECTIVE: To investigate the regulatory effects of deoxyribozyme on the expression of Period 1 gene in vitro and on the morphine-induced psychic dependence in mice. METHODS: The specific deoxyribozymes toward Period 1 mRNA was designed by MFold analysis and synethsized chemically. By LipofectAMINE mediated DNA transfection technique, DRz164 and pcDNA3-Per1 were introduced into NIH3T3 cells. The effects of deoxyribozyme on Period 1 gene were studied by reverse transcript-polymerase chain reaction (RT-PCR) and flow cytometry(FCM). The morphine-induced reward in mice was observed in a conditioned place preference test after pretreatment of the mice with the intracerebroventricular administration of deoxyribozyme. RESULTS: After NIH3T3 cells were transfected by pcDNA3-Per1 and DRz164, the Period 1 mRNA was reduced by 42.4%. And PERIOD proteins were decreased by 57.5%. After being pretreated with deoxyribozyme, the mice did not show morphine-induced place preference. CONCLUSION: DRz164 can highly block the expression of Period 1 gene, which cleaves the Period 1 mRNA in the transfected cells specifically. The suppression of morphine-induced place preference can be effected by pretreating the mice with alleviating their psychic dependence on morphine.

[Design and application of biological rhythm automatic monitor and data analysis system].

OBJECTIVE: To design and develop a biological rhythm monitor and data analysis system for studying biological rhythm in animals under different conditions. METHODS: The system was a distributed digital control system consisting of a computer and zeitgeber generators. Acquisition of data on animal activity and generation of zeitgeber were modularized to meet the multiple requirements of experiment. System functions and animal adaptability to this system were detected by monitoring the rhythm of mouse activities under different light-dark cycles. RESULT: The animal experiment showed that the functions of this system, including data acquisition, data communication, light regulation and data analysis, were qualified for application. There was no apparent malfunction. CONCLUSION: The system is safe, reliable and easy to operate. The system could be a very useful system to study biological rhythm in animals.

[Effects of different light-dark cycles on total and differential leukocyte counts as well as spontaneous locomotor activity in mice].

OBJECTIVE: To investigate the effect of different light-dark cycles on total and differential counts of leukocyte as well as spontaneous locomotor activity in mice. METHOD: 144 ICR mice were raised under different light-dark cycles including LD 7 h/7 h, LD 12 h/12 h and LD 16 h/16 h for 8 weeks. Blood samples were obtained by enucleation of eye for total and differential counts of leukocyte. Another 9 mice were also divided into 3 groups and fed under same conditions as above. Their activities were recorded continuously. RESULT: The change of spontaneous locomotor activity and leukocyte count were synchronized with the light-dark cycle. CONCLUSION: Different light-dark cycles can influence not only the spontaneous locomotor activity, but also total and differential leukocyte counts in mice.

[The influence of circadian gene mPeriod2 on Lewis cancer cell].

OBJECTIVE: To investigate the role of circadian gene mPeriod2 (mPer2) on tumor proliferation and apoptosis. METHOD: The eukaryotic mPer2 expression vector (pcDNA 3.1-mPer2) based on pcDNA 3.1 was transfected into the cultured Lewis tumor cell by liposome method in vivo. The expression of mPer2 in transfected Lewis tumor cell was detected by immunohistochemistry and flowcytometry. The stable transfected cell line was screened by G418 and its proliferation and apoptosis was examined by flowcytometry. RESULT: Expression of PERIOD2 protein in pcDNA 3.1-mPer2 transfected Lewis tumor cell was proved by immunohistochemistry and flowcytometry. Flowcytometry result of mPer2 expression Lewis tumor cell showed less proliferation and high rate of apoptosis. CONCLUSION: mPer2 expression can suppress the proliferation of tumor cell and increase the apoptosis of it.

[Cleavage of the deoxyribozyme targeting Period1 mRNA in vitro and its effects on morphine-induced reward in mice].

OBJECTIVE: To study the cleavage of the deoxyribozyme targeting Period1 (Per1) mRNA in vitro and its effect on the opiate-induced reward in mice. METHOD: The specific deoxyribozyme (DRz164) targeting Per1 mRNA was designed after MFold analysis and being synthesized chemically. The cleavage reactions containing DRz164 and Per1 RNA fragments transcripted in vitro were performed under certain conditions. DIG Nucleic Acid Detection Kit was used to detect the cleavage activity of DRz164. The morphine-induced reward in mice was observed in a conditioned place preference test after pretreatment with DRz164. RESULT: When DRz164 and Per1 RNA transcripts were incubated under certain conditions with 37 degrees C for 30, 60, 90, and 120 min, about 36.4%, 40.5%, 47.8%, 63% of Per1 RNA transcripts were cleaved by DRz164 respectively. After pretreated with the deoxyribozyme, mice didn't show a morphine-induced place preference. CONCLUSION: DRz164 targeting per1 mRNA has the specific cleavage activity which increases with the time in vitro. Suppression of morphine-induced place preference occurs by pretreatment with the deoxyribozyme. Mice show a lack of morphine-induced reward, which alleviates the psychic dependence on the morphine.

[The effect of ribozyme specially cleaving per1 mRNA on c-fos mRNA and its expression in hippocampus of morphine addicted mice].

OBJECTIVE: To study the change of c-fos mRNA and protein in hippocampus of morphine addicted mice after injected with ribozyme specially cleaving per1 mRNA. METHOD: The recombined plasmid pcDNA 3.1-per1RZ DNA was injected into the ventricles of morphine addicted mice to transcript the corresponding ribozyme which cleaves per1 mRNA particularly. And then, the brains of mice were fixed by perfusion. The level of c-fos mRNA was assayed by in situ hybridization and c-fos protein was detected by immunohistochemical staining. RESULT: The level of c-fos mRNA and protein decreased after injection of the recombined plasmid pcDNA 3.1-per1RZ DNA expressing the ribozyme cleaving per1 mRNA. CONCLUSION: The ribozyme specially cleaving per1 mRNA has potential function in inhibiting the transcription and expression of c-fos and blocking the morphine addiction.

[Effects of different light-dark cycle on learning and memory in mice].

OBJECTIVE: To study the effect of different light-dark cycles on learning and memory in mice. METHOD: Seventy-two ICR mice were raised under different light-dark cycles including LD 5h/5h, LD 12h/12h and LD 22h/22h for 6 weeks. The locomotor activity was recorded continuously. Morris water-maze task was used as the judging criteria for spatial learning and memory. RESULT: The locomotor activity rhythm was consistent with the light-dark cycle. The period of light-dark cycle shorter than 24 h such as 10 h could effect on the ability of learning and memory in mice. CONCLUSION: The short period of light-dark cycle can improve the ability of learning and memory in mice.

[The role of circadian gene period1 in morphine reward in mice].

OBJECTIVE: To investigate the role of circadian gene Period1 on drug dependence. METHOD: The ribozyme cleave the Period1 mRNA (per1RZ) was designed and the per1RZ expression vector (pcDNA 3.1-per1RZ) was constructed based on eukaryotic expression vector pcDNA 3.1. In vitro cleavage experiment proved the target cleave Period1 mRNA ability of per1RZ. Conditional place preference (CPP) paradigm used to investigate the effect of intracerebroventricular (ICV) injection of pcDNA 3.1-per1RZ on drug reward in BALB/C mice. RESULT: Quantitative analysis of in vitro cleavage experiment showed the efficacy of ribozyme per1RZ, about 60% of the Period1 mRNA was cleaved by ribozyme per1RZ. CPP test displayed the block of drug reward in pcDNA 3.1-per1RZ ICV injection group. Period1 expression in brain was attenuated of pcDNA 3.1-per1RZ ICV injection group demonstrated by western blot. CONCLUSION: Interfere the Period1 expression in brain could attenuate the psychological dependence of drug in mammals.