Cytotoxic Effects of Xanthohumol and Its Combination with Cisplatin on Human Metastatic Lung Cancer H1299 Cells

Aims:Non-small cell lung cancer (NSCLC) accounts for high lung cancer death that is mostly associated with advanced disease stage at diagnosis and resistance to chemotherapy. In the present study, we investigated whether xanthohumol, a prenylated flavonoid of hop plant, induces metastatic lung cancer H1299 cell death, and whether in combination with cisplatin there are additive effects. Methodology:H1299 cells were grown and treated with xanthohumol (6.25, 12.5, or 25 μM), cisplatin (12.5, 25, or 50 μM) and the combination of cisplatin and xanthohumol for 24 h. Cell viability, cell morphology, chromatin condensation, ɣH2AX, cPARP-1, capsase-3, p21WAF1/CIP1and p14ARFgenes were analyzed Results:Xanthohumol, cisplatin, and the combination of cisplatin and xanthohumol inhibited H1299 cells viability. Cisplatin growth inhibitory effects were potentiated by xanthohumol. Xanthohumol induced chromatin condensation and apoptosis and potentiated cisplatin’s effect vs cisplatin alone. Further investigation of growth inhibitory effects, xanthohumol alone induced γH2AX foci formation and the combination potentiated γH2AX foci formation. Cisplatin, xanthohumol at 25 μM, and the combination of cisplatin and xanthohumol at 6.25 and 12.5 μM increased cPARP-1 level. Active caspase-3 was increased by cisplatin, 12.5 μM of xanthohumol, and the combination of xanthohumol and cisplatin. Xanthohumol at 6.25 or 12.5 μM potentiated cisplatin effect on active caspase-3 and cPARP-1, respectively. Xanthohumol at 25 μM significtly induced the expression cell cycle control genes p21WAF1/CIP1and p14ARF. These results indicate that xanthohumol inhibits proliferation of H1299 cells and induces cell death through cleavage of PARP-1 and activation of caspase-3. The combination of cisplatin and xanthohumol potentiated cytotoxic effects of each other compound.Conclusion:The present study suggests that xanthohumol poses apoptotic effects and potentiates cisplatin’s growth inhibitory effects on metastatic lung cancer cells

A study on high function binding site of HDAC1 in p21 WAF1/CIP1 promoter region in breast cancer MCF-7 cells / 中国药理学通报

Aim Toinvestigatethespecificbinding sites that HDAC1 can be recruited to regulate the tran-scriptional activity of p21 WAF1/CIP1 promoter in the breast cancer MCF-7 cells.Methods ThebreastcancerMCF-7 cells in logarithmic growth phase were starved with FBS free medium for 24 hours,and treated with 20 μmol·L-1 SAHA(S group)or 0. 625 nmol·L-1 Leptin(L group)for 24 hours,and the cells that were cultured in the complete RPMI 1640 medium without any treatment were assigned as control group (B group).The DNA-ChIP was followed the manufactur-er′s protocol for the assay.The cell lysis was prepared and incubated with anti-HDAC1 antibody overnight at 4℃.DNA fragments binding anti-HDAC1 antibody were gathered and purified.The relative expression level of DNA fragments from TSS to the upstream of the p21 WAF1/CIP1 promoter region(+2 ~-4000 bp)bind-ing with antibody was detected by Real-time PCR and analyzedby2-ΔΔCTmethod.Results InBgroup, HDAC1 had high affinity with the f1 and f 8 fragmentsof p21 WAF1/CIP1 promoter compared to the other fragemts,and showed the highest affinity with the f8 fragment.In S group,the binding ability of HDAC1 to the f1 ~f10 fragment of p21 WAF1/CIP1 promoter was sig-nificantly lower than that of the control.The binding activity of HDAC1 to f8 fragment was the lowest,while reversing to reach the peak after leptin treatment.Con-clusions HDAC1canberecruitedtop21WAF1/CIP1pro-moter by the cell proliferation signal during the prolifer-ation of breast cancer MCF-7 cells.The DNA fragment from -2800 to -3200 bp in the upstream of p21 WAF1/CIP1 promoter is the target functional region for the binding with HDAC1 .

High-affinity Binding Sites for Estrogen Receptor α in the p21WAF1/CIP1 Promoter Region in Breast Cancer MCF-7 Cells / 中国医科大学学报

Objective To investigate the specific sites that estrogen receptor (ER)α could be recruited to in the p21WAF1/CIP1 promoter region to regulate its transcriptional activity in MCF-7 cells,and to clarify the molecular mechanism of suberoylanilide hydroxamic acid (SAHA) and leptin in the regulation of p21WAFI/CIP1 promoter function.Methods MCF-7 cells were starved by culturing them in fetal calf serum-free medium for 24 hours,and then treated with 20 μmol/L of 0.88 μL SAHA (SAHA group) or 0.625 nmol/L of 10 μL leptin (leptin group) for 24 hours,or cultured in complete RPMI-1640 medium (control group).Cell lysates were incubated with anti-ERα antibody for ChIP analysis.The relative expression levels of DNA fragments,ranging from the TSS to upstream of the p21WAF1/CIP1 promoter region (+2 to-4 000 bp),that bound the antibody were detected by real-time PCR.Results In the control group,the relative expression levels of f1,f2,and f8 DNA fragments that bound the antiERα antibody were two-fold higher than the relative expression of the f9 fragment (P ＜ 0.01).In the SAHA and leptin groups,the relative expression of f1 to f10 DNA fragment that bound anti-ERα antibody was significantly lower than that of the control.The binding affinity of ERα for the f8 fragment was the lowest (P ＜ 0.01) in the SAHA group,and it was significantly lower than that in the leptin group (P ＜ 0.01).Conclusion ERα could be recruited to the p21WAFI/CIP1 promoter via signaling pathways activated during the proliferation of breast cancer MCF-7 cells.Moreover,the DNA fragment ranging from-2 800 to-3 200 bp upstream of the p21 WAF1/CIP1 promoter is the target functional region for high-affinity binding with ERα.

Synergistic effect of ERα on HDAC1 regulatingtranscription of p21WAF1/CIP1 in breast cancer MCF-7 cells / 中国药理学通报

Aim To investigate the specific binding sites that histone deacetylases 1(HDAC1) and estrogen receptor α(ERα)can be recruited to regulate the transcriptional activity of p21WAF1/CIP1 promoter in the breast cancer MCF-7 cells, and to clarify the molecular mechanism of suberoylanilide hydroxamic acid(SAHA) and leptin regulating p21WAF1/CIP1 promoter function.Methods The breast cancer MCF-7 cells in logarithmic growth phase were starved with FBS free medium for 24 hours, and treated with 20 μmol·L-1 SAHA(SAHA group) or 0.625 nmol·L-1 leptin(Leptin group) for 24 hours.The cells that were cultured in complete RPMI 1640 medium without any treatment were assigned as control group(Basal group).The cell lysis was prepared and incubated respectively with anti-HDAC1 and anti-ERα antibody by chromatin-immunoprecipitation(ChIP) method overnight at 4℃.The DNA-ChIP was followed the manufacturer′s protocol for the assay.DNA fragments binding anti-HDAC1 and anti-ERα antibody were gathered and purified.The relative expression level of DNA fragments from TSS to the upstream of the p21WAF1/CIP1 promoter region(+2~-4 000 bp) binding with antibody was detected by real-time PCR and analyzed by 2-△△CT method.Results In basal group, HDAC1 and ERα had high affinity with the f1 and f8 fragments of p21WAF1/CIP1 promoter compared to the f4 fragment.In SAHA group, the binding ability of HDAC1 and ERα to the f1 and f8 fragments of p21WAF1/CIP1 promoter was significantly lower than that of the control, while reversing to reach the peak after leptin treatment.Conclusions HDAC1 and ERα can be recruited to p21WAF1/CIP1 promoter by the cell proliferation signal during the proliferation of breast cancer MCF-7 cells.The DNA f1(from 0 to-400 bp) and f8(from-2 800 to-3 200 bp) fragment in the upstream of p21WAF1/CIP1 promoter are the target functional region for the binding with HDAC1and ERα.

SAHA affects cell cycle of MCF-7 breast cancer cells byregulating acetylated levels of p21WAF1/CIP1 promoter / 中国药理学通报

Aim To study the regulation mechanisms of deacetylase inhibitor SAHA in p21WAF1/CIP1 promoter acetylation in breast cancer MCF-7 cells.Methods We used quantitative real-time PCR, Western blot and DNA-ChIP to determine the effects on the regulation of cell cycle with SAHA treatment in MCF-7 cells.By DNA-ChIP, we assessed the acetylation level of p21WAF1/CIP1 promoter.Results SAHA significantly affected the expression of cell cycle-related factors, and induced the mRNA and protein expression of p21WAF1/CIP1.SAHA could adjust the acetylation level of p21WAF1/CIP1 promoter.Conclusion SAHA regulates the cell cycle progression by adjusting the acetylation level of p21WAF1/CIP1 promoter in MCF-7 cells.

p21WAF1/CIP and Regulation of Breast Cancer Cell Cycle / 沈阳医学院学报

Abnormal proliferation is a key point for the occurrence and development of breast cancer. p21WAF1/CIP is essential to maintain a normal growth and development of the organisms, and its dysfunction can lead to organism overgrowth. The function of p21WAF1/CIP occurs mainly in post?transcriptional and transcriptional level. Especially, the transcription regulation of p21WAF1/CIP is very important to cell cycle progression of breast cancer via epigenetic change in histone deacetylation.

Expression of Plk1, Cyclin B1 and p21WAF1 in cervical carcinoma and their clinical signifi-cance / 临床与实验病理学杂志

Purpose To investigate the expression of polo-like kinase 1 (Plk1) and Cyclin B1, p21WAF1in cervical carcinoma, and to determine the relationship between the expression of the three proteins and tumor clinicopathological features. Methods The expres-sion of Plk1, Cyclin B1 and p21WAF1 was detected in 102 cases of cervical carcinoma, 20 cases of (cervical intraepithelial neoplasia, CIN) , and 20 cases of nomal cervical tissues by the technique of tissue chip and immunohistochemical staining of EliVision. Statistical analyses of the data were performed with SPSS 19. 0 software. Results The positive rates of Plk1 in cervical carcinoma and CIN were 70. 5%, 55. 0%, respectively, which were significantly higher than normal cervical tissues (10%) (P<0. 01);The positive rates of Cyclin B1 in cervical carcinoma and CIN were 52. 9% and 30. 0%, respectively, which were significantly higher than normal cervical tissues (10%)(P <0.01); The positive rates of p21WAF1 in cervical carcinoma and CIN were 23.5% and 10.0%, respectively, which were significantly higher than normal cervical tissues ( 0 ) ( P<0. 01 ) . There were no significant differences between cervical carcinoma and CIN in the positive rates of Plk1, Cyclin B1 and p21WAF1. The expression of Plk1 was associated with the depth of carci-noma invasion (P<0. 05), that of Cyclin B1 was associated with lymph node metastases and the depth of carcinoma invasion (P<0. 05)and that of p21WAF1 in cervical carcinoma was associated with histological grade (P<0. 05). In cervical carcinoma, the expres-sion of Plk1 was positively correlated with Cyclin B1 (rs =0. 297, P=0. 002) and negatively correlated with p21WAF1(rs = -0. 403, P<0. 001). Conclusion The expression of Plk1, Cyclin B1 and p21WAF1 is involved in the occurrence and development of cervical carcinoma, and the former two are also related with prognosis of cervical carcinoma. The combination of the three would provide a new target for clinical treatment.

Lentivirus-mediated p21/Waf1 silencing enhances replicative potential of bladder cancer-specific oncolytic adenovirus and its inhibition effect on proliferation of EJ cells / 肿瘤

Objective: To investigate the effects of p21/Waf1 gene silencing on replicative potential of bladder cancer-specific oncolytic adenovirus and the proliferation inhibition of EJ cells. Methods: The shRNA targeting p21/Waf1 gene (p21/Waf1-shNA) was designed and synthesized, then the annealing oligonucleotide fragments were subcloned into pMagic 7.1 vector containing coding gene of green fluorescent protein (GFP) to construct recombinant lentiviral plasmid pLVT1051, which was confirmed by PCR and DNA sequencing. The 293T cells were co-transfected with three plasmids including pLVT1051, pCMV-VSV-G and pCMV-dR8.91 to produce the recombinant lentivirus. The EJ cells were infected with recombinant lentivirus carrying p21/Waf1-shRNA, and the puromycin was used to screen out the stably infected EJ cells. The expression levels of p21/Waf1 mRNA and protein in EJ cells after infection with recombinant lentivirus carrying p21/Waf1-shRNA were detected by real-time fluorescent quantitative-PCR and Western blotting, respectively. The p21/Waf1 gene-silenced EJ cells were infected with bladder cancer-specific oncolytic adenovirus Ad/PSCAE/UPII/E1A, then the expression levels of virus replication-related proteins E1A and Hexon were detected by Western blotting, and the cytotoxic effect on EJ cells was examined by MTT method. Results: Recombinant lentiviral vector carrying p21/Waf1-shRNA targeting p21/Waf1 gene was successfully established and confirmed by DNA sequencing. The recombinant lentivirus was harvested. The stably infected EJ cells were successfully screened out by using puromycin forr two weeks. The expression levels of p21/Waf1 mRNA and protein in EJ cells infected with recombinant lentivirus carrying p21/Waf1-shRNA were significantly reduced (both P < 0.05). The expression levels of E1A and Hexon proteins in p21/Waf1 gene-silenced EJ cells infected with bladder cancer-specific oncolytic adenovirus Ad/PSCAE/UPII/E1A were up-regulated, and the proliferation inhibition of EJ cells was enhanced (P < 0.01). Conclusion: The p21/Waf1 gene-silenced EJ cells are successfully constructed, and p21/Waf1 gene silencing can enhance the replicative ability of bladder cancer-specific oncolytic adenovirus and significantly inhibit the proliferation of EJ cells.

Cellular senescence is regulated by p21Waf1/Cip1 methylation / 重庆医学

Objective To explore the effect of p21Waf1/Cip1 methylation changes on the process of cellular senescence .Methods Bisulfite sequencing was used to analyze the methylation changes of p21Waf1/Cip1 in the process of cellular senescence;p21Waf1/Cip1 ex‐pression was detected by RT‐PCR and Western‐blot ;Middle‐aged 2BS cells was treated by 5‐aza‐CdR and cellular senescence was detected by MTT and SA‐β‐Gal staining .Results Bisulfite sequencing analysis of p21Waf1/Cip1 promoter showed that CpGs were methylated by 1 .25% in the young 2BS cells ,by 27 .27% in the middle‐aged 2BS cells ,while only by 0 .64% in the senescent cells . The expression of p21Waf1/Cip1 was low in the young(28 PD) 2BS cells ,it increased first(35 PD) but decreased later in the middle‐aged(42 PD) cells .In the senescent 2BS cells ,p21Waf1/Cip1 expression was further increased .5‐aza‐CdR treatment resulted in de‐creased growth rate but increasedβ‐Gal staining of middle‐aged 2BS cells .Conclusion The process of cellular senescence is regula‐ted by the status of p21Waf1/Cip1 methylation ,and p21Waf1/Cip1 demethylation accelerates cellular senescence .

Antitumor Activity of Erythromycin on Human Neuroblastoma Cell Line (SH-SY5Y) / 华中科技大学学报（医学）（英德文版）

Antitumor effects of erythromycin and the related mechanism were investigated in the present study.Neuroblastoma cells (SH-SY5Y) were exposed to erythromycin at different concentrations for different durations.Cell proliferation was measured by cell counting,and cell viability was examined by MTT assay.Cell cycle phase distribution and the cytosolic calcium level were detected by flow cytometry.Mitochondrial membrane potential was measured by the JC-1 probe staining and fluorescent microscopy.The expression of an oncogene (c-Myc) and a tumor suppressor [p21 (WAF1/Cipl)] proteins was analyzed by using Western blotting.Erythromycin could inhibit the proliferation of SH-SY5Y cells in a concentration- and time-dependent manner.The cell cycle was arrested at S phase.Mitochon drial membrane potential collapsed and the cytosolic calcium was overloaded in SH-SY5Y cells when treated with erythromycin.The expression of c-Myc protein was down-regulated,while that of p21 (WAF1/Cip1) protein was up-regulated.It was concluded that erythromycin could restrain the proliferation of SH-SY5Y cells.The antitumor mechanism of erythromycin might involve regulating the expression ofc-Myc and p21 (WAF1/Cip1) proteins.

Clinical significance and expression of Skp2, p27 and p21 in Ovarian Serous Cystadenocarcinomas / 中国综合临床

ObjectiveTo investigate the correlation of Skp2,p27kiP1 and p21WAF1 expression with the clinicopathological features of ovarian serous cystadenocarcinomas.Methods Expressions of Skp2 ,p27kiP1 and p21WAF1 were examined by immunohistochemical staining in 124 epithelial ovarian tumors (25 serous cystadenomas, 19 borderline serous cystadenomas, and 80 serous cystadenocarcinomas) Results(1) The expression of Skp2 in serous cystadenocarcinomas (47.5％)was significantly higher than that in borderline serous cystadenomas (0％)and serous cystadenomas (0％)(P ＜ 0.001) .The p27kiP1 expression in serous cystadenocarcinomas (35.0％) was significantly lower than that in borderline serous cystadenomas(73.7％)and serous cystadenomas (80.0％) .The p21WAF1 staining frequency in serous cystadenocarcinomas (38.8％)was significantly lower than in borderline serous cystadenomas (73.7％)and serous cystadenomas (80.0％) .(2) The Skp2 protein expression in serous cystadenocarcinomas was positively correlated with clinicopathological stage,histological differentiation degree and lymph node metastasis of the tumors.The p27kiP1, p21WAF1 protein expression in serous cystadenocarcinomas was reversely correlated with clinicopathological stage and histological differentiation degree of the tumors(Ps ＜ 0.05) .(3) The Skp2 protein expression in serous cystadenocarcinomas was reversely correlated with that of p27kiP1 , p21WAF1.Conclusion The Skp2 protein expression in serous cystadenocarcinomas was increased and positively correlated with the clinicopathological features of ovarian serous cystadenocarcinomas.Skp2 protein expression was reversely correlated with p27kip1 ,p21WAF1.Skp2 protein expression may play an important role in the development and progression of serous cystadenocarcinomas.

Anti-proliferative effect of artesunate on MDA-MB-231 cell and its mechanisms / 中华内分泌外科杂志

Objective To study the effects of artesunate(ART)on estrogen receptor negative breast cancer cell line MDA-MB-231 and its mechanisms.Methods Treated with ART for 3 days,MDA-MB-231 cells proliferation was examined by MTT assay.The morphological and uhrastructural changes of MDA-MB-231 cells were observed under microscope and electronic microscope.Immunocytochemistry was used to detect the expression of Bax,mn23,Bcl-2,and P21 WAFl/CIPl.Results ART treatment led to a dose dependent inhibition of MDA-MB-231 cells.ART could change the morphology and ultrastructure of MDA-MB-231 cens.After treatment with ART(2μmol/L)for 72 hours,immunocytochemical staining showed that the expression of Bax,nm23,and P21WAF1/CIP1 was upregulated in comparison to the control group(P0.05).Conclusions ART shows an anti-proliferative effect on MDA-MB-231 cells.The mechanisms may be related to upregnlation of Bax,nm23 and P21WAF1/CIP1 expression.

Vaccinia virus regulates expression of p21WAF1/Cip1 in A431 cells

In this paper, we provide evidence that both the mRNA and protein levels of the cyclin-dependent kinase (CDK) inhibitor p21WAF1/CDK-interacting protein 1 (Cip1) increase upon infection of A431 cells with Vaccinia virus (VACV). In addition, the VACV growth factor (VGF) seems to be required for the gene expression because infection carried out with the mutant virus VACV-VGF- revealed that this strain was unable to stimulate its transcription. Our findings are also consistent with the notion that the VGF-mediated change in p21WAF1/Cip1 expression is dependent on tyrosine kinase pathway(s) and is partially dependent on mitogen-activated protein kinase/extracellular-signal regulated kinase 1/2. We believe that these pathways are biologically significant because VACV replication and dissemination was drastically affected when the infection was carried out in the presence of the relevant pharmacological inhibitors.

Relation between expression of CDX2 and p21~(WAF1) protein in colorectal cancer and TCM deficiency syndrome / 国际中医中药杂志

Objective To study the expression of CDX2 and p21~(WAF1) protein in colorectal cancer and its correlation with the progression of colorectal cancer., and to explore the eorrelativity of CDX2 and p21~(WAF1) genes expression with TCM deficiency Syndrome. Methods Immunohistochemical method was used to detect the expressions of CDX2 and p21~(WAF1) in45 patients of colorectal carcinoma tissues and peri-cancerous tissues, who received the treatment at the People's Hospital of Jiuquan City between April, 2000 and August, 2005. According to TCM syndrome differentiation, the patients were divided into sthenia syndrome group and asthenia syndrome group and sthenia-ashenia syndrome group. Analyze the Correlation between CDX2 and p21~(WAF1) expression and TCM syndrome. Results The positive rate of CDX2 and p21~(WAF1) was 86.7% and 35.6% respectively in colorectal carcinoma tissues. There was no correlation between CDX2 and p21~(WAF1) in colorectal cancer tissues. The positive rate of p21~(WAF1) was different in three groups, which was much lower in the asthenia syndrome group (11.76%) than the sthenia syndrome group (41.67%)and sthenia-asthenia syndrome group (56.25%). Conclusion CDX2 and p21~(WAF1) positive rate was different in cancer tissue and resection margin. The two genes may be associated with colorectal cancer development. CDX2 was not correlated with p21~(WAF1). The lower expression of p21~(WAF1) indicates poor prognosis. The positive rate of p21~(WAF1) was different between deficiency syndrome and excess syndrome, p21~(WAF1) is probably a marker to determine the asthenia and sthenia syndrome. Asthenia syndrome indicates a poor prognosis.

Ginsenoside-Rg1 protects human dermal fibroblasts against 8-methoxypsoralen plus ultraviolet A-induced photoaging through p53-dependent signaling pathways / 中国药理学通报

Aim To investigate the roles of p53-dependent signaling pathways in the process of ginsenoside Rg1 protection against 8-methoxypsoralen(8-MOP) and subsequent ultraviolet A(UVA) irradiation induced photoaging model in human dermal fibroblasts(HDFs).Methods Photoaging model was established by 8-MOP/UVA in skin HDFs.Flow cytometry, enzyme cytochemistry, immunofluorescence and Western blot were employed.Results Pretreatment with ginsenoside Rg1 could significantly reduce the amount of UVA-generated 8-oxo-dG and relieve the photoaging representation.Compared with 8-MOP/UVA treatment group, pretreatment with Ginsenoside Rg1 could decrease the expression of SA β-galatosides(SA-β-Gal), and down-regulate the level of senescence associated proteins(p16 and p21).Conclusions Ginsenoside Rg1 has prominent dose-dependent antagonism on senescence of skin HDFs induced by 8-MOP/UVA, and its mechanism may be due to its antioxidation which reduces the production of photoproducts to protect telomere against abnormal shortening.

Estrogen exerts the effect on stress-induced senescence of vascular smooth muscl cells in vitro / 解剖学报

Objective To explore the effects of estrogen on stress-induced senescence of vascular smooth muscle cells (VSMCs) and the underlying mechanisms. Methods The VSMCs of passage 2-3 cultured from female SD rats were induced into senescence by exposing to 150μmol/L H2O2 in the presence or absence of different concentrations(10-10mol/L-10-8mol/L) of 17β-estradiol (E2). The expressions or activities of senescence associated marker DcR2, senescence-associated beta-galactosidase (SA-β-Gal), oncogene Ras and p21WAF1 were detected by flow cytometry, cytochemical staining, pull-down assay or Western blotting analysis. Results Flow cytometry analysis showed that in the physiological concentrations, E2 significantly inhibited the H2O2-promoted high-level expression of DcR2 of VSMCs in a dose-dependent manner, with a highest inhibitive rate at 14.48%±0.6%(E2=10-8 mol/L;P<0.05, n =3);this inhibitive effect could be blocked by a E2 receptors inhibitor ICI 182,780. Cytochemistry staining showed that the rate of SA-β-Gal positive VSMCs induced by H2O2 decreased in presence of 10-8mol/L E2 (20.5%±1.4% vs 9.6%±0.9%;P<0.05, n =9). Pull-down assay and Western blotting analysis revealed that administration of 10-8mol/L E2 obviously reduced the H2O2-induced activity of Ras (0.60±0.06 vs 0.26±0.04;P<0.05, n =3) and expression of p21WAF1 (0.46±0.04 vs 0.33±0.02;P<0.05, n =3). Conclusion E2 exerts, an inhibitive effects on stress-induced senescence of VSMCs by suppressing the activity of Ras and expression of p21WAF1. This finding suggests a novel mechanism for the hormone's anti-atheroschlerotic effects.

Cigarette Smoke Extract Inhibits the Proliferation of Alveolar Epithelial Cells and Augments the Expression of P21WAF1 / 华中科技大学学报（医学）（英德文版）

Cigarette smoking is intimately related with the development of chronic obstructive pulmonary diseases, and alveolar epithelium is a major target for the exposure of cigarette smoke ex- tract. In order to investigate the effect of cigarette smoke extract on the proliferation of alveolar epithelial cell type Ⅱand its relationship with P21WAF1, the alveolar epithelial type Ⅱ cell line (A549) cells were chosen as surrogate cells to represent alveolar epithelial type Ⅱ cells. MTT assay was used to detect cell viability after interfered with different concentrations of cigarette smoke ex-tract. It was observed cigarette smoke extract inhibited the growth of A549 cells in a dose- and time-dependent manner. The morphological changes, involving the condensation and margination of nuclear chromatin, even karyorrhexis, were observed by both Hoechst staining and electronic mi-croscopy. Flow cytometry analysis demonstrated the increased cell percentages in G1 and subG1phases after the cells were incubated with cigarette smoke extract. The expression of p21WAF1 protein and mRNA was also significantly increased as detected by the methods of Western blot or reverse transcription-polymerase chain reaction respectively. In conclusion, cigarette smoke extract inhibits the proliferation of alveolar epithelial cell type Ⅱ and blocks them in G1/S phase. The intracellular accumulation of P21WAF1 may be one of the mechanisms which contribute to cigarette smoke ex-tract-induced inhibition of cell proliferation.

Expression and significance of HSG/Mfn2 and P21 WAF1 in non-small cell Lung cancer / 中国综合临床

Objective To investigate the expression of hyperplasia suppressor gene(HSG)/mitofusin 2 (Mfn2)and P21 WAF1 in non-small cell lung cancer(NSCLC).Methods The expression of(HSG/Mfn2)and P21 WAF1 was detected in 92 cases of NSCLC samples by immunohistochemistry(SP).Results The absorptance value of the expression of HSG/Mfn2 in squamous cell carcinoma,adenocarcinoma,and non-cancer tissue were 15.06±2.73,12.21±2.96 and 10.36±3.60,respectively(P<0.05),and they were associated with tumor differentiation.The absorptanee valtue of the expression of P21 WAF1 in squamous cell carcinoma,adenocarcinoma,and non-cancer tissue were 3.16±0.98,3.44±0.22,0.06±0.32.The expression of P21 WAF1 in squamous cell carcinoma and adenocarcinoma Was higher than that in non-cancer tissue(P<0.05),and was closely associated with tumor differ-entiation and lymph node metastasis.Conclusion HSG/Mfn2,P21WAF1 is closely related to the pathogenesis and development of lung cancer.There is positive correlation between the HSG/Mfn2 and P2l WAF1 in lung cancer.

Expression of Mutant p53 Protein, p21(waf1/cip1) and Cyclin D1 in Dysplasia and Adenocarcinoma of Stomach / 대한소화기내시경학회지

BACKGROUND/AIMS: Gastric carcinoma is a major cause of morbidity and mortality in Korea. It evolves through dysplasia to an invasive adenocarcinoma. The carcinogenesis of dysplasia and adenocarcinoma in the stomach was investigated by examining the levels of mutant p53 protein, p21(waf1/cip1), and cyclin D1 expression in gastric dysplasia and invasive adenocarcinoma. METHODS: Formalin- fixed paraffin-embedded tumors were examined immunohistochemically using the monoclonal antibodies to the 53 protein, p21(waf1/cip1) and cyclin D1. RESULTS: Mutant p53 protein, p21(waf1/cip1) and cyclin D1 expression were found in 66.6% (12/18), 72.2% (13/18) and 33.8% (6/18) of dysplasia, and 45.0% (9/20), 15.0% (3/20) and 30.0% (6/20) of invasive adenocarcinoma, respectively. CONCLUSIONS: These results suggest that p21(waf1/cip1), which is controlled by the p53 protein, plays a more important role in the carcinogenesis of the stomach than cyclin D1.

Clinical Analysis According to p21(Waf1/Cip1) and p27(Kip1) Expression in Gastric Cancer

PURPOSE: The p21(Waf1/Cip1) protein inhibits the cell cycle by inhibiting the phosphorylation at the G1-->S check point, and the p27(Kip1) protein similarly performs the suppressor function by controlling the p27-mediated G1 arrest. In this study, we analysed the clinical status and survival rates in correlations with p21 and p27 expression patterns in gastric cancer. MATERIALS AND METHODS: Between 1993 and 1997, 192 patients who underwent surgeries in Catholic Medical Center were analysed retrospectively in this study. Immunohistochemical staining was performed and if the nuclei of the tumor cells were stained, we assumed those as positive results. Statistical analysis was based on clinicopathological findings and differences in survival rates. RESULTS: The expression rate of p27 was 28.1% and 15.6% in p21 each. The ratio of T1-2(80.0%) was significantly high in p21 (+), but the ratio of T3-4 (50.6%) was slightly high in p21 (-). There was no statistical significance regarding other factors. The results in p27 was not much different from expression rate of p21 in T-stage. In addition, p27 expression in diffuse type (91.3%) was higher than in intestinal type (62.7%) by Lauren's classification (P <0.05). Also, there was no statistical significance in other factors. In the correlation of p21 and p27, p27 was positive when p21 was positive (53.5%). Conversely, p27 was negative when p21 was negative (76.5%, p <0.05). In the p21 and p27 combination test, there was higher rate of T1-2 (87.5%) in p21 (+)/p27 (+), and higher rate of T3-4 (58.1%) in p21 (-)/p27 (-) (P <0.05). Results showed higher rate of intestinal type (100%) in p21 (+)/p27 (+), and diffuse type (87.0%) was dominant in p21 (-)/p27 (-) (P <0.05) by Lauren's classification. Moreover, there was no statistical significance in the 5-year survival rate in the expression of p21 and p27, and the 5-year survival rate was highest in the case of p21 (+)/p27 (+) without statistical significance. CONCLUSION: In our study, p21(Waf1/Cip1) and p27(Kip1) expressed similar patterns. The expression of p21(Waf1/Cip1) and p27(Kip1) affected the degree of invasiveness of the tumor, and. Combined examination result revealed the correlation of p21(Waf1/Cip1) and p27(Kip1) with Lauren's classification and depth of invasion of the tumor. However, we assumed that little difference between the survival rates depending on expression of p21(Waf1/Cip1) and p27(Kip1) has limited their value as predictable prognostic indicators.