Krill oil ameliorates benign prostatic hyperplasia by regulating G1-phase cell cycle arrest and altering signaling pathways and benign prostatic hyperplasia-associated markers

Krill oil(KO)exhibits various biological activities,such as anti-inflammatory and antitumor effects.However,the inhibitory effects of benign prostatic hyperplasia(BPH)in vitro and in vivo have not yet been studied.This study investigated the anti-BPH effects of KO extracted by an enzymatic hydrolysis method.KO treatment inhibited the proliferation of WMPY-1 and BPH-1 cells by induction of G0/G1 phase arrest through the modulation of positive and negative regulators in both prostate cell types.KO treatment stimulated phosphorylation of c-Jun N-terminal kinase(JNK)and p38 signaling.In addition,KO changed the expression of BPH-related markers(5α-reductase,androgen receptor,FGF,Bcl-2,and Bax)and the activity of the proliferation-mediated NF-κB binding motif.KO-induced levels of proliferation-mediated molecules of prostate cells were attenuated in the presence of siRNA-specific p-38(si-p38)and JNK(si-JNK).Furthermore,the administration of KO alleviated prostate size and weight and the cell layer thickness of prostate glands in a testosterone enanthate-induced BPH rat model.KO treatment altered the level of dihydrotestosterone in serum and the expression levels of BPH-related markers in prostate tissues.Finally,KO-mediated inhibition of prostatic growth was validated by histological analysis.These results suggest that KO has an inhibitory effect on BPH in prostate cells in vitro and in vivo.Thus,KO might be a potential prophylactic or therapeutic agent for patients with BPH.

CARP-1(Cell Division Cycle and Apoptosis Regulator Protein 1)的生物功能和表达模式探究

本文对CARP-1生物功能和表达模式进行了研究,结果表明,作为一个关键的细胞内复合物,传感激活细胞的增殖或凋亡信号通路,在经典Wnt通路中,CARP-1与β-catenin功能共激活,协助β-catenin在Wnt目标基因作用中的转录激活,涉及到一些蛋白信号的扩散和转移;参与caspase泛素化蛋白酶途径,与APC/C复合,在CFM-4的阻碍作用下,阻滞G2/M细胞周期;与P53、P160复合,刺激靶细胞激素代谢,调控细胞周期,通过增强CDK1和P21的水平来调节细胞凋亡。

TuBG1 promotes hepatocellular carcinoma via ATR/P53-apoptosis and cycling pathways

Background:As reported,γ-tubulin(TuBG1)is related to the occurrence and development of various types of malignant tumors.However,its role in hepatocellular cancer(HCC)is not clear.The present study was to investigate the relationship between TuBG1 and clinical parameters and survival in HCC patients.Methods:The correlation between TuBG1 and clinical parameters and survival in HCC patients was ex-plored by bioinformatics analysis.Immunohistochemistry was used for the verification.The molecular function of TuBG1 was measured using colony formation,scratch assay,trans-well assay and flow cytometry.Gene set enrichment analysis(GSEA)was used to pick up the enriched pathways,followed by investigating the target pathways using Western blotting.The tumor-immune system interactions and drug bank database(TISIDB)was used to evaluate TuBG1 and immunity.Based on the TuBG1-related immune genes,a prognostic model was constructed and was further validated internally and externally.Results:The bioinformatic analysis found high expressed TuBG1 in HCC tissue,which was confirmed us-ing immunohistochemistry and Western blotting.After silencing the TuBG1 in HCC cell lines,more G1 arrested cells were found,cell proliferation and invasion were inhibited,and apoptosis was promoted.Furthermore,the silence of TuBG1 increased the expressions of Ataxia-Telangiectasia and Rad-3(ATR),phospho-P38 mitogen-activated protein kinase(P-P38MAPK),phospho-P53(P-P53),B-cell lymphoma-2 associated X protein(Bax),cleaved caspase 3 and P21;decreased the expressions of B-cell lymphoma-2(Bcl-2),cyclin D1,cyclin E2,cyclin-dependent kinase 2(CDK2)and CDK4.The correlation analysis of immunohistochemistry and clinical parameters and survival data revealed that TuBG1 was negatively corre-lated with the overall survival.The constructed immune prognosis model could effectively evaluate the prognosis.Conclusions:The increased expression of TuBG1 in HCC is associated with poor prognosis,which might be involved in the occurrence and development of HCC.

Intrinsic apoptotic pathway and G2/M cell cycle arrest involved in tubeimoside I-induced EC109 cell death

Objective： Squamous esophageal carcinoma is highly prevalent in developing countries, especially in China. Tu Bei Mu （TBM）, a traditional folk medicine, has been used to treat esophageal squamous cell carcinoma （ESCC） for a long term. tubeimoside I （TBMS1） is the main component of TBM, exhibiting great anticancer potential. In this study, we investigated the mechanism of TBMS1 cytotoxic effect on EC109 cells. Methods： Comparative nuclear proteomic approach was applied in the current study and we identified several altered protein spots. Further biochemical studies were carried out to detect the mitochondrial membrane potential, cell cycle and corresponding proteins＇ expression and location. Results： Subcellular proteomic study in the nucleus from EC109 cells revealed that altered proteins were associated with mitochondrial function and cell proliferation. Further biochemical studies showed that TBMSl-induced molecular events were related to mitochondria-induced intrinsic apoptosis and P21-cyclin B 1/cdc2 complex-related G2/M cell cycle arrest. Conclusions： Considering the conventional application of TBM in esophageal cancer, TBMS1 therefore may have a great potential as a chemotherapeutic drug candidate for ESCC.

Overexpression of p27^(KIP1)induced cell cycle arrest in G_1 phase and subsequent apoptosis in HCC-9204 cell line

AIM We have previously reported that inducible over-expresaion of Bak may prolong cell cycle in G1 phase and lead to apoptosis in HCC-9204 cells. This study is to investigate whether p27KIP1 plays an important role in this process. MEHODS In order to elucidate the exact function of p27KIP1 in this process, a zinc inducible p27KIP1 stable transfectant and transient p27KIP1- GFP fusion transfectant were constructed. The effects of inducible p27KIP1 on cell growth, cell cycle arrest and apoptosis were examined in the mock, control pMD vector, and pMD-KIP1 transfected HCC-9204 cells. RESULTS This p27KIP1-GFP transfectant may transiently express the fusion gene. The cell growth was reduced by 35% at 48 h of p27KIP1 induction with zinc treatment as determined by trypan blue exclusion assay. These differences remained the same after 72 h of p27KIP1 expression, p27KIP1 caused cell cycle arrest after 24 h of induction, with 40% increase in G1 population. Prolonged p27KIP1 expression in this cell line induced apoptotic cell death reflected by TUNEL assay. Fourty-eight h and 72 h of p27KIP1 expression showed a characteristic DNA ladder on agarose gel electrophoresis.

Paris chinensis dioscin induces G2/M cell cycle arrest and apoptosis in human gastric cancer SGC-7901 cells

AIM:To investigate the anti-tumor effects of Paris chinensis dioscin(PCD)and mechanisms regarding cell cycle regulation and apoptosis in human gastric cancer SGC-7901 cells.METHODS:Cell viability was analyzed by the 3-(4,5-di-methylthiazol-2-yl)-2,5-diphenyl-tetrazolium bromide assay.Cell apoptosis was evaluated by flow cytometry and laser scanning confocal microscope(LSCM)using Annexin-V/propidium iodide(PI)staining,and the cell cycle was evaluated using PI staining with flow cytom-etry.Intracellular calcium ions were detected under fluorescence microscope.The expression of cell cycle and apoptosis-related proteins cyclin B1,CDK1,cytochrome C and caspase-3 was measured by immunohistochemical staining.RESULTS:PCD had an anti-proliferation effect on human gastric cancer SGC-7901 cells in a dose-and time-de-pendent manner.After treatment of SGC-7901 cells with PCD,apoptosis appeared in SGC-7901 cells.Morpho-logical changes typical of apoptosis were also observed with LSCM by Annexin V/PI staining,and the cell number of the G0/G1 phase was decreased,while the number of cells in the G2/M phase was increased.Cell cycle-related proteins,such as cyclin B1 and CDK1,were all down-regulated,but caspase-3 and cytochrome C were up-regulated.Moreover,intracellular calcium accumulation occurred in PCD-treated cells.CONCLUSION:G2/M phase arrest and apoptosis induced by PCD are associated with the inhibition of CDK-activating kinase activity and the activation of Ca2+-related mitochondrion pathway in SGC-7901 cells.

Effect of p27^(KIP1) on cell cycle and apoptosis in gastric cancer cells

AIM： To elucidate the effect of p27^KIP1 on cell cycle and apoptosis regulation in gastric carcinoma cells. METHODS： The whole length of p27^KIP1 cDNA was transfected into human gastric cancer cell line SCG7901 by lipofectamine. Expression of p27^KIP1 protein or mRNA was analyzed by Western blot and RNA dot blotting, respectively. Effect of p27^KIP1 on cell growth was observed by MTT assay and anchorage-independent growth in soft agar. Tumorigenicity in nude mice was used to assess the in vivo biological effect of p27^KIP1. Flow cytometry, TUNEL, and electron microscopy were used to assess the effect of p27^KIP1 on cell cycle and apoptosis. RESULTS： Expression of p27^KIP1 protein or mRNA increased evidently in SCG7901 cells transfected with p27^KIP1. The cell growth was reduced by 31% at 48 h after induction with zinc determined by cell viability assay. The alteration of cell malignant phenotype was evidently indicated by the loss of anchorage-independent growth ability in soft agar. The tumorigenicity in nude mice was reduced evidently （0.55±0.14 cm vs 1.36±0.13cm, P〈0.01）. p27^KIP1 overexpression caused cell arrest with 36% increase （from 33.7% to 69.3%, P〈0.01） in G1 population. Prolonged p27^KIP1 expression induced apoptotic cell death reflected by pre-G1 peak in the histogram of FACS, which was also confirmed by TUNEL assay and electron microscopy. CONCLUSION： p27^KIP1 can prolong cell cycle in G1 phase and lead to apoptosis, p27^KIP1 may be a good candidate for cancer gene therapy.

The role of NBS1 in DNA double strand break repair, telomere stability, and cell cycle checkpoint control

The genomes of eukaryotic cells are under continuous assault by environmental agents and endogenous metabolic byproducts. Damage induced in DNA usually leads to a cascade of cellular events, the DNA damage response. Failure of the DNA damage response can lead to development of malignancy by reducing the efficiency and fidelity of DNA repair. The NBS1 protein is a component of the MRE11/RAD50/NBS 1 complex （MRN） that plays a critical role in the cellular response to DNA damage and the maintenance of chromosomal integrity. Mutations in the NBS1 gene are responsible for Nijmegen breakage syndrome （NBS）, a hereditary disorder that imparts an increased predisposition to development of malignancy. The phenotypic characteristics of cells isolated from NBS patients point to a deficiency in the repair of DNA double strand breaks. Here, we review the current knowledge of the role of NBS1 in the DNA damage response. Emphasis is placed on the role of NBS1 in the DNA double strand repair, modulation of the DNA damage sensing and signaling, cell cycle checkpoint control and maintenance oftelomere stability.

Effect of cis-9,trans-11-conjugated linoleic acid on cell cycle of gastric adenocarcinoma cell line(SGC-7901)

AIM: To determine the effect of cis -9, trans -11-conjugated linoleic acid (c9, t11-CLA) on the cell cycle of gastric cancer cells (SGC-7901) and its possible mechanism in inhibition cancer growth. METHODS: Using cell culture and immunocytochemical techniques, we examined the cell growth, DNA synthesis, expression of PCNA, cyclin A, B(1), D(1), p16(ink4a) and p21(cip/waf1) of SGC-7901 cells which were treated with various c9, t11-CLA concentrations (25, 50, 100 and 200 micromol.L(-1))of c 9, t 11-CLA for 24 and 48h, with a negative control (0.1% ethane). RESULTS: The cell growth and DNA synthesis of SGC-7901 cells were inhibited by c9, t11-CLA.SGC-7901 cells. Eight day after treatment with various concentrations of c9, t11-CLA mentioned above, the inhibition rates were 5.92%, 20.15%, 75.61% and 82.44%, respectively and inhibitory effect of c9, t11-CLA on DNA synthesis (except for 25 micromol.L, 24h) showed significantly less (3)H-TdR incorporation than that in the negative controls (P【0.05 and P【0.01). Immunocytochemical staining demonstrated that SGC-7901 cells preincubated in media supplemented with different c9, t11-CLA concentrations at various times significantly decreased the expressions of PCNA (the expression rates were 7.2-3.0%, 24h and 9.1-0.9% at 48h, respectively), Cyclin A (11.0-2.3%, 24h and 8.5-0.5%,48h), B(1) (4.8-1.8% at 24h and 5.5-0.6% at 48h)and D(1) (3.6-1.4% at 24h and 3.7%-0 at 48h) as compared with those in the negative controls(the expressions of PCNA, Cyclin A, B(1) and D(1) were 6.5% at 24h and 9.0% at 48h, 4.2% at 24h and 5.1% at 48h, 9.5% at 24h and 6.0% at 48h,respectively)(P【0.01), whereas the expressions of P16(ink4a) and P21(cip/waf1), cyclin-dependent kinases inhibitors(CDKI), were increased. CONCLUSION: The cell growth and proliferation of SGC-7901 cell is inhibited by c9, t11-CLA via blocking the cell cycle, with reduced expressions of cyclin A,B(1) and D(1) and enhanced expressions of CDKI(P16(ink4a) and p21(cip/waf1)).

Dexamethasone suppresses DU145 cell proliferation and cell cycle through inhibition of the extracellular signal-regulated kinase 1 /2 pathway and cyclin D1 expression

Aim： To determine the mechanisms of glucocorticoids in inhibiting advanced prostate cancer growth. Methods： The cell proliferation and cell cycle of prostate cancer DU145 cells following dexamethasone treatment were determined by proliferation assay and fluorescence-activated cell sorter. Western blot analysis was carried out to evaluate the effects of dexamethasone on phosphorylation of extracellular signal-regulated kinase （ERK）1/2 and expression of cyclin D1 in DU145 cells with or without glucocorticoid receptor （GR） antagonist RU486. Reverse transcription- polymerase chain reaction verified the expression of GR mRNA in DU145 cells. Results： Dexamethasone significantly inhibited DU 145 cell proliferation at the G0/G1 phase. Westem blot analysis showed a dramatic reduction of ERK1/2 activity and cyclin D1 expression in dexamethasone-treated cells. The decreased phosphorylation of ERK1/2 in dexamethasone-treated cells was attenuated by GR blockade. Additionally, the effects of dexamethasone in inhibiting cyclin D1 expression were altered by GR blockade. Conclusion： Dexamethasone suppresses DU145 cell proliferation and cell cycle, and the underlying mechanisms are through the inhibition of phosphorylation of ERK1/2 and cyclin D1 expression. The inhibition of ERK1/2 phosphorylation and cyclin D1 expression is attenuated by GR blockade, suggesting that GR regulates ERK1/2 and cyclin D1 pathways. These observations suggest that dexamethasone has a potential clinical application in prostate cancer therapy.

Adhesion of different cell cycle human hepatoma cells to endothelial cells and roles of integrin β_1

AIM: To investigate the adhesive mechanical properties of different cell cycle human hepatoma cells (SMMC-7721) to human umbilical vein endothelial cells (ECV-304), expression of adhesive molecule integrinβ1 in SMMC-7721 cells and its contribution to this adhesive course. METHODS: Adhesive force of SMMC-7721 cells to endothelial cells was measured using micropipette aspiration technique. Synchronous G1 and S phase SMMC-7721 cells were achieved by thymine-2-deoxyriboside and colchicines sequential blockage method and double thymine-2-deoxyriboside blockage method, respectively. Synchronous rates of SMMC-7721 cells and expression of integrinβ1 in SMMC-7721 cells were detected by flow cytometer. RESULTS: The percentage of cell cycle phases of general SMMC-7721 cells was 11.01% in G2/M phases, 53.51% in G0/G1 phase, and 35.48% in S phase. The synchronous rates of G1 and S phase SMMC-7721 cells amounted to 74.09% and 98.29%, respectively. The adhesive force of SMMC-7721 cells to endothelial cells changed with the variations of adhesive time and presented behavior characteristics of adhesion and de-adhesion. S phase SMMC-7721 cells had higher adhesive forces than d phase cells [(307.65±92.10)×10-10N vs(195.42±60.72)×10-10N, P<0.01]. The expressive fluorescent intensity of integrinβ1 in G1 phase SMMC-7721 cells was depressed more significantly than the values of S phase and general SMMC-7721cells. The contribution of adhesive integrinβ1 was about 53% in this adhesive course. CONCLUSION: SMMC-7721 cells can be synchronized preferably in d and S phases with thymine-2-deoxyriboside and colchicines. The adhesive molecule integrinβ1 expresses a high level in SMMC-7721 cells and shows differences in various cell cycles, suggesting integrin β1 plays an important role in adhesion to endothelial cells. The change of adhesive forces in different cell cycle SMMC-7721 cells indicates that S phase cells play predominant roles possibly while they interact with endothelial cells.

MicroRNA-15a-cell division cycle 42 signaling pathway in pathogenesis of pediatric inflammatory bowel disease

AIM To determine whether cell division cycle(Cdc)42 is regulated by microRNA(miR)-15 a in the development of pediatric inflammatory bowel disease(IBD).METHODS We cultured 293 T cells, used plasmids and performed dual-luciferase assay to determine whether Cdc42 is a miR-15 a target gene. We cultured Caco-2 cells, and stimulated them with tumor necrosis factor(TNF)-α. We then employed lentiviruses to alter the expression of miR-15 a and Cdc42. We performed quantitative reverse transcription polymerase chain reaction(qRTPCR) and immunofluorescence to determine whether Cdc42 is regulated by miR-15 a in Caco-2 cells. Finally, we collected ileocecal tissue by endoscopy from patients and performed qRT-PCR to examine the expression of miR-15 a and Cdc42 in pediatric IBD patients.RESULTS Target Scan and dual-luciferase assay revealed thatCdc42 was a miR-15 a target gene. MiR-15 a expression increased(P = 0.0038) and Cdc42 expression decreased(P = 0.0013) in cells stimulated with TNF-α, and the expression of the epithelial junction proteins zona occludens(ZO)-1(P < 0.05) and E-cadherin(P < 0.001) decreased. Cdc42 levels decreased in miR-15 a-mimic cells(P < 0.001) and increased in miR-15 a inhibitor cells(P < 0.05). ZO-1 and E-cadherin decreased in mi R-15 a-mimic cells(P < 0.001) but not in the miR-15 a inhibitor + TNF-α cells. In Lv-Cdc42 + TNF-α cells, ZO-1 and E-cadherin expression increased compared to the Lv-Cdc42-NC + TNF-α(P < 0.05) or miR-15 a-mimic cells(P < 0.05). Fifty-four pediatric IBD patients were included in this study, 21 in the control group, 19 in the Crohn's disease(CD) active(AC) group, seven in the CD remission(RE) group, and seven in the ulcerative colitis(UC) group. MiR-15 a increased and Cdc42 decreased in the CD AC group compared to the control group(P < 0.05). miR-15 a decreased and Cdc42 increased in the CD RE group compared to the CD AC group(P < 0.05). miR-15 a was positively correlated with the Pediatric Crohn's disease Activity Index(PCDAI)(P = 0.006), while Cdc42 was negatively correlated with PCDAI(P = 0.0008). Finally, miR-15 a expression negatively correlated with Cdc42 in pediatric IBD patients(P = 0.0045).CONCLUSION Mi R-15 a negatively regulates epithelial junctions through Cdc42 in Caco-2 cells and pediatric IBD patients.

Ethanol extract of Kalopanax septemlobus leaf inhibits HepG2 human hepatocellular carcinoma cell proliferation via inducing cell cycle arrest at G_1 phase

Objective:To investigate the effects of an ethanol extract of Kalopanax septemlobus(Thunb.)Koidz.leaf(EEKS) on cell proliferation in human hepatocellular carcinoma cells and its mechanisms of action.Methods:Cells were treated with EEKS and subsequently analyzed for cell proliferation and flow cytometry analysis.Expressions of cell cycle regulators were determined by reverse transcriptase polymerase chain reaction analysis and Western blotting,and activation of eyclin-associaled kinases studied using kinase assays.Results:The EEKS suppressed cell proliferation in both HepG2 and Hep3 B cells,but showed a more sensitive anli-proliferative activity in HepG2 cells.Flow cytometry analysis revealed an association between the growth inhibitory effect of EEKS and with G_1 phase cell cycle arrest in HepG2 cells,along with the dephosphorylation of retinoblastoma protein(pRB) and enhanced binding of pRB with the E2 F transcription factor family proteins.Treatment with EEKS also increased the expression of cyclin-dependent kinase(CDK) inhibitors,such as p21WAF1/CIP1 and p27KIP1.without any noticeable changes in G_1 cyclins and CDKs(except for a slight decrease in CDK4).Treatment of HepG2 cells with EEKS also increased the binding of p21 and p27 with CDK4 and CDK6.which was paralleled by a marked decrease in the cyclin D- and cyclin E-associated kinase activities.Conclusions:Overall,our findings suggest that EEKS may be an effective treatment for liver cancer through suppression of cancer cell proliferation via G_1,cell cycle arrest Further studies arc required to identify the active compounds in EEKS.

Cell cycle exit and neuronal differentiation 1-engineered embryonic neural stem cells enhance neuronal differentiation and neurobehavioral recovery after experimental traumatic brain injury

Our previous study showed that cell cycle exit and neuronal differentiation 1(CEND1)may participate in neural stem cell cycle exit and oriented differentiation.However,whether CEND1-transfected neural stem cells can improve the prognosis of traumatic brain injury remained unclear.In this study,we performed quantitative proteomic analysis and found that after traumatic brain injury,CEND1 expression was downregulated in mouse brain tissue.Three days after traumatic brain injury,we transplanted CEND1-transfected neural stem cells into the area surrounding the injury site.We found that at 5 weeks after traumatic brain injury,transplantation of CEND1-transfected neural stem cells markedly alleviated brain atrophy and greatly improved neurological function.In vivo and in vitro results indicate that CEND1 overexpression inhibited the proliferation of neural stem cells,but significantly promoted their neuronal differentiation.Additionally,CEND1 overexpression reduced protein levels of Notch1 and cyclin D1,but increased levels of p21 in CEND1-transfected neural stem cells.Treatment with CEND1-transfected neural stem cells was superior to similar treatment without CEND1 transfection.These findings suggest that transplantation of CEND1-transfected neural stem cells is a promising cell therapy for traumatic brain injury.This study was approved by the Animal Ethics Committee of the School of Biomedical Engineering of Shanghai Jiao Tong University,China(approval No.2016034)on November 25,2016.

6-OHDA Induces Cycle Reentry and Apoptosis of PC12 Cells through Activation of ERK1/2 Signaling Pathway

This study investigated the effect and mechanism of cell cycle reentry induced by 6-hydrodopamine （6-OHDA） in PC12 cells. By using neural differentiated PC12 cells treated with 6-OHDA, the apoptosis model of dopaminergic neurons was established. Cell viability was measured by MTT. Cell apoptosis and the distribution of cell cycle were assessed by flow cytometry. Western blot was used to detect the activation of extracellular regulator kinasel/2 （ERK1/2） pathway and the phosphorylation of retinoblastoma protein （RB）. Our results showed that after PC12 cells were treated wtih 6-OHDA, the viability of PC12 cells was declined in a concentration-dependent manner. Flow cytornetry revealed that 6-OHDA could increase the apoptosis ratio of PC12 cells in a time-dependent manner. The percentage of ceils in G0/G1 phase of cell cycle was decreased and that in S phase and G2/M phase increased. Simultaneously, ERK1/2 pathway was activated and phosphorylated RB increased. It was concluded that 6-OHDA could induce cell cycle reentry of dopaminergic neurons through the activation of ERK1/2 pathway and RB phosphorylation. The aberrant cell cycle reentry contributes to the apoptosis of dopaminergic neurons.

Effect of Exogenous p16ink4a and hRb1 Genes on Cell Cycle Regulation of Osteosarcoma Cell

To study the effect on regulation of cell cycle of osteosarcoma cell line MG63 tranceduced with exogenous p16ink4a and hRbl genes, pIRES-p16ink4a-hRb1, pIRES-p16ink4a and pIREShRbl plasmids were constructed by gene recombination technology. The recombinant plasmid was transferred into osteosarcoma cell line MG63 by metafectene, and the resistant clones were selected by G418 selective medium, mRNA and protein expression of osteosarcoma cell line were assayed by RT-PCR and Western-Blot respectively. Cell cycle and apoptosis were analyzed by subG1 flow cytometric. Cell proliferation was tested by MTT. In the genome of these transfected target cells, the expression of p16ink4a and hRb1 mRNA and protein were detected respectively in vitro. It was demonstrated with subG1 flow cytometric analysis and MTT method that p16ink4a and hRbl genes cooperation more significantly inhibited cell growth and induced a more marked G1 arrest and apoptosis than p16ink4a/hRb1 alone （P〈0.01）. Coexpression of exogenous p16ink4a with hRbl broke the regulatory feedback loop of p16ink4a-cyclinD1/CDK-hRbl and played a more significant role in inhibiting cell growth as well as inducing cell apoptosis than p16ink4a or hRbl did alone in vitro.

Vitamin C Inhibits Benzo[a]pyrene-Induced Cell Cycle Changes Partly via Cyclin D1/E2F Pathway in Human Embryo Lung Fibroblasts

Objective To study the molecular mechanism of the inhibitory effects of vitamin C on benzo[a]pyrene （B[a]P）-induced changes of cell cycle in human embryo lung fibroblast （HELF） cells. Methods The stable transfectants, HELF transfected with antisense cyclin D1 and antisense CDK4, were established. Cells were cultured and pretreated with vitamin C before stimulation with B[a]P for 24 h. The expression levels of cyclin DI, CDK4, E2FI, and E2F4 were determined by Western blot. Flow cytometric analysis was employed to detect the distributions of cell cycle. Results B[a]P significantly elevated the expression levels of cyclin D 1, E2F1, and E2F4 in HELF cells. Vitamin C decreased the expression levels of cyclin D 1, E2F1, and E2F4 in B [a]P-stimulated HELF cells. Dose-dependent relationships were not found between the different concentrations of vitamin C （10, 100, 500, 1000, and 5000 lamol/L） and the expression levels of cyclin D 1, E2F1, and E2F4 in HELF cells. The expression levels of cyclin D1, E2FI, and E2F4 in B[a]P-treated transfectants were lower than those in B[a]P-treated HELF cells. The expression levels of cyclin DI and E2F4 treated with vitamin C and antisense cyclin D1 were decreased compared with those treated with antisense cyclin DI alone. The effects of vitamin C combined with antisense CDK4 on the expression levels of cyclin DI and E2FI/E2F4 were similar to those of antisense CDK4 alone. B[a]P progressed HELF cells from GI to S phase. Both vitamin C and antisense cyclin DI suppressed the changes of cell cycle progressed by B[a]P. However, antisense CDK4 did not attenuate the above changes. Vitamin C combined with antisense CDK4 markedly suppressed B[a]P-induced changes of cell cycle as compared with antisense CDK4. But the inhibitory effects of vitamin C combined with antisense cyclin DI on B[a]P-induced changes of cell cycle were similar to those of vitamin C alone or antisense cyclin DI alone. Conclusions B[a]P progressed HELF cells from G1 to S phase via intracellular signaling pathway of cyclin D I/E2F. Vitamin C may modulate this signaling pathway to protect cells from injury caused by B[a]P.

Role of the Na^+/K^+/2Cl^- cotransporter NKCC1 in cell cycle progression in human esophageal squamous cell carcinoma

AIM: To investigate the role of Na+/K+/2Cl- cotransporter 1 (NKCC1) in the regulation of genes involved in cell cycle progression and the clinicopathological significance of its expression in esophageal squamous cell carcinoma (ESCC).

Influence of CDK1 and CDK2 siRNA interference on tumor cell cycle and cell apoptosis

Objective： We investigated the influence of CDK1 and CDK2 expression inhibited by cotransfection of CDK1 and CDK2 siRNA on cell cycle and apoptosis, explored the exact role of cell cycle master regulator in tumor cell apoptosis process. Methods： The siRNA targeting the CDK1 and CDK2 genes were synthesized and simultaneously cotransfected into Hela cells by lipofectamine 2000.48 or 60 h after the cotransfection, CDK1 and CDK2 protein expressions were examined by Western blot. Cell cycle distribution was analyzed by flow cytometry. Cell apoptosis was detected by the Annexin V/PI method. The changes of the transfected cell morphological under a microscope after Wright-Giemsa Staining were studied. Results： CDK1 and CDK2 protein expression was decreased at 48 or 60 h after cotransfection. The accumulation of the G2/M and S phase population in cell cycle of the cotransfected cells at 48 or 60 h after transfection was enhanced obviously compared with control. The ratio of apoptotic cell of cotransfected cells at 48 or 60 h after transfection was increased significantly compared with control. More binucleate or multinucleate ceJls among cotransfected cells were observed under the microscope. Conclusion： The decreased expression of CDK1 and CDK2 by cotransfection of CDK1 and CDK2 siRNA not only leads to tumor cell cycle arrest in S phase and G2/M phase, but also induces tumor cell apoptosis.

Expression of p27Kip1, A Cell Cycle Repressor Protein with Dual Roles for Both Cancer Prevention and Promotion, Is Regulated Primarily at the Level of Unusual p27Kip1 mRNA—A Short Concept Proposal

The p27Kip1 is a cell cycle repressor protein that regulates primarily the cell cycle transition from G1 to S phase and hence the DNA replication is in the S phase and cell division in the M phase. Expression of p27Kip1 protein has dual roles for both cancer prevention and promotion. For example, numerous nutritional and chemopreventive anti-cancer agents specifically increase the expression of p27Kip1 protein without directly affecting the expression of any other cell cycle regulatory proteins. On the other hand, pro-cancer agents (like glucose, insulin and other growth factors frequently seen in obesity and/or diabetes) specifically decrease the expression of p27Kip1 protein without directly affecting the expression of any other cell cycle regulatory proteins. Unlike expression of any other cell cycle regulatory proteins, expression of p27Kip1 protein is very unusual. The mRNA of p27Kip1 has a very long and unusual 5’-untranslated region (from -575 to -1 in human). It appears that the 5’-untranslated region of p27Kip1 mRNA forms two alternative secondary structures. One increases the expression of p27Kip1 protein when anti-cancer agents are added and another decrease the expression of p27K1p1 when pro-cancer agents are added. For this short concept proposal, Dr. Albert Einstein’s “visualized thought experiments (German: Gedanken experiment)” were used as a fundamental tool for understanding how either anti- or pro-cancer agents bring the primary structure of the 5’-untranslated region of p27Kip1 mRNA into two alternative secondary structures, thereby either increasing or decreasing, respectively, the translation initiation of p27Kip1 protein.