The Role Of Clock Gene NPAS2in Breast Carcinogenesis And The Effect Of Acanthopanax Senticosus Flavone On Expression Of Clock Gene And Its Anticancer Mechanism

(1) The expression and clinical significance of clock gene Npas2in human breastcarcinomaObjective: To investigate the expression of clock gene Npas2in breast cancer anddiscuss its relationship with the carcinogenesis and development of breast cancerMethod: The expression of Npas2was analized by immunohistochemistry, including30cases of breast infiltrating carcinoma,20cases of nomal breast tissue,20cases of breastprecancerous change(10cases of lobular carcinoma in situ and10cases of intraductalpapilloma) and20cases of breast adenofibroma. Breast infiltrating carcinoma andprecancerous change were devided into4stages according to the standard of UICC:10casesin T0stage,9cases in I stage,16cases in II stage and5cases in III stage;11of which hadlymphonode metastasis,19of which were negative lymphonode metastasis. Motic ImagesAdvanced3.2image analysator was used to measure gray scale under200multiplemicroscope for5vision fields. The expression of Npas2was indicated by the gray scale. Theless of the gray scale, the more of the expression of Npas2; the more of the gray scale, the lessof the expression of Npas2.Result: The Npas2protein could be expressed in nomal breast tissue, breastprecancerous change and infiltrating carcinoma. Specific color was in intracytoplasm asbrown drop. The Npas2was expressed positively in breast nomal tissue with the gray scale(114.3±2.31), the expression was decreased obviously in infiltrating carcinoma with the grayscale(159.5±6.79). The Npas2was expressed positively in breast precancerous change withthe gray scale(132.3±2.11), the expression decreased obviously in breast adenofibroma withthe gray scale(206.3±3.02); Npas2were depressed consecuently in filtrating carcinoma of I, IIand III stage with the gray scale (142.3±2.46),(161.2±4.22) and (176.3±2.93). Npas2was lessexpressed in stage II and III comparing with stage T0and I, and the difference of them hadstatistical significance (P<0.05). In breast infiltrating carcinoma, the gray scales in positivelymphonode metastasis and negative lymphonode metastasis were (156.8±3.23) and (166.3±4.04), the differnence of them had no statistical significance(P>0.05), which might beresulting from the insufficient samples.Conclusion: Npas2was less depressed obviously in breast cancer, which was associatedwith the carcinogenesis, pathology and clinical stage. The low expression of Npas2wasrelated to the tumor size and infiltrating depth, high expression of Npas2played a protectiverole in the development of breast cancer. The high expression of Npas2in breast precancerouschange indicated that it might have an effect in prohibiting the canceration. Low function ofNpas2might act as an important factor in breast carcinogenesis. (2) The effect of Acanthopanax senticosus flavone in expression of Npas2and Per2inMCF-7cell line and its anticancer mechanismObjective: To detect the role of Acanthopanax senticosus flavone(ASF) in theexpression of Npas2and Per2, growth depressant effect, cell proliferation, expression ofapoptosis associated protein and stability of the genome in MCF-7cell, and then, to discussthe effect of ASF in regulating circadian rhythm and its anticancer mechanism.Method: The depressant effect of different density of ASF in MCF-7cell growth wasdetected by MTT; The effect of Acanthopanax senticosus flavone in MCF-7cell cycle wasdetected by flow cytometry; Real time PCR was used to detect the effect of ASF in expressionof cell cycle associated protein Cyclin D1and P21in different density and at different time;immunofluorescence stain was used to detect MCF-7cell and the expression of clock geneNpas2and Per2after giving ASF, the fluorescence expressive intensity of Npas2and Per2could be quantified by software. Tunel cell apoptosis detected method was used to observe theapoptotic induced effect of ASF in different densities in MCF-7cell, apoptotic index wasmanifested by the percentage of positive apoptosis cell in the samevision field.immunofluorescence stain was used to detect the effect of ASF in the expression of apoptoticassociated protein such as Bax,bcl-2and c-myc in MCF-7cell. The change of transcriptionallevel in Brg1was detected by real time PCR. Western Blotting was used to detect the proteinexpression of pRb and phosphorylated pRb at6h,12h and24h after giving ASF. Protein strappray scale was scanned by image analysis, quantity of dephosphorylated pRb was calculatedas quantity of total pRb substract phosphorylated pRb, semiquantitative analysis could bedone by ppRb/pRb and(pRb-ppRb)/pRb. Result: ASF with density of100μmol/L could increase the expression of Npas2and Per2in MCF-7cell obviously, and inhibit the proliferation of MCF-7cell, the effect manifesteddose and time dependence. It didn't have the inhibition function to MCF-7when the densitywas below75μmol/L after24h, and even the inhibition was even weaker at the lower density,which promoted the cell proliferation. The dentisy that could inhibit cell growth was75μmol/L after48h. ASF with density of100μmol/L could increase the cells in G1stage,decrease the cells in S stage and G2/M stage, which manifested that ASF could prevent thecell growing from G1to S stage, and also prevent cell getting into the dividing phase toinhibit the caryomitosis, which indicated ASF could inhibit the tumor cell growth by changingcell cycle. After given ASF with density of50μmol/L for24h, Cyclin D1genetic transcriptionincreased obviously and it increased dramatically when the density of which was100μmol/L,and the time was6h in advance, it dropped to normal after12h. P21genetic transcription hadno relationship with ASF, and the transcription increased at6h, its expression decreased in12-24h. Apoptosis could be seen occasionally in normal MCF-7cell, the apoptotic index was0.8%after giving ASF with density of50μmol/L, the apoptotic index was5.2%with thedensity of100μmol/L, and it could reach12.6%with the density of200μmol/L. It could bemanifested that ASF with density of200μmol/L could induce cell apoptosis(p<0.01), theapoptotic index increased with the density of ASF. Quantified with fluorescence expressiveintensity after giving ASF with density of100μmol/L, the expression of Bax increased whilethe expression of Bcl-2decreased obviously(p<0.01), the expression of c-myc decreasedeither. ASF with density of50μmol/L and100μmol/L could enhance the Brg1genetictranscription in6h, the expression reached its peak in12h(p<0.01), and dropped below normalin24h. After given ASF with density of100μmol/L in MCF-7cell for12h and24h, theexpression of pRb phosphorylated protein decreased and dephosphorylated protein increased,it manifested a statistic difference (p<0.05).Conculusion:1. ASF with density of100μmol/L could increase the protein expressionof Npas2and Per2in MCF-7cell, which might act as the mechanism of circadian rhythmregulation and its anticancer role.2. ASF with density of150μmol/L could inhibit the MCF-7cell proliferation in vitro by changing the cell cycle and regulating the expression of cell cycleprotein Cyclin D1and P21.3. ASF with density of200μmol/L could induce cell apoptosis bydown regulating apoptotic inhibiting gene Bcl-2and up regulating apoptotic promotive geneBax to play its anticancer role.4. ASF with density of100μmol/L could induce the geneexpressions of Brg1, Npas2and Per2in MCF-7, which indicated that clock gene Npas2and Per2and anti-oncogene Brg1could play an important role in breast carcinogenesis. ASF withdensity of100μmol/L could make pRB in low phosphorylation in MCF-7cell and arrest thecell cycle by regulating the cell cycle control point to help the cell recovery and stabilize theDNA, which indicated that ASF with density of100μmol/L could play an important role ingene stabilization.